Methods and compositions for treating cancers and enhancing therapeutic immunity by selectively reducing immunomodulatory M2 monocytes

ABSTRACT

The present disclosure provides pharmaceutical compositions comprising nucleic acids capable of targeting IGF-1R expression in M2 cells. The present disclosure also provides methods for the selective reduction of M2 cells by targeting expression of IGF-1R in these cells. The present disclosure further provides methods for treating cancer and enhancing therapeutic by targeting expression of IGF-1R in M2 cells in patients. The pharmaceutical composition of the present invention is effective when administered systemically to subjects in need thereof. The ease of administration of the pharmaceutical composition facilitates treatment and enhances patient compliance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/867,000, filed Jan. 10, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/095,877, filed Apr. 11, 2016, which claims thebenefit of U.S. Provisional Application No. 62/145,758, filed Apr. 10,2015, the disclosures of which are hereby incorporated by reference intheir entirety.

FIELD OF INVENTION

The present disclosure relates to methods and compositions for treatingcancers and enhancing therapeutic immunity by selectively reducing M2cells by targeting these cells with antisense nucleic acids directedagainst Insulin-like Growth Factor 1 Receptor (IGF-1R).

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: a computer readableformat copy of the sequence listing (filename:205961_5000_03US_SequenceListing.txt).

BACKGROUND

Monocytes are a type of white blood cell that originate from myeloidprogenitors in bone marrow. From there they enter the peripheral bloodstream and later migrate into tissues. In the tissues, after exposure tolocal growth factors, pro-inflammatory cytokines, and microbialcompounds, monocytes differentiate into macrophages and dendritic cells.Macrophages derived from monocyte precursors undergo specificdifferentiation into the classically polarized (M1) macrophages and thenon-classically activated (M2) macrophages. Normally, macrophages servethree main functions in the immune system. These are phagocytosis,antigen presentation, and cytokine presentation. In addition, certaintypes of cancers (such as, for example, breast cancer, astrocytoma, headand neck squamous cell cancer, papillary renal cell carcinoma Type II,lung cancer, pancreatic cancer, gall bladder cancer, rectal cancer,glioma, classical Hodgkin's lymphoma, ovarian cancer, and colorectalcancer) exhibit elevated levels of M2-like macrophages within the tumorand similar M2 monocytes circulating in the periphery. Despite advancesin cancer therapy, the prognosis for these cancers remains poor andattempts to treat these cancers using conventional treatments such as,for example, chemotherapy, external beam radiation, and brachytherapyhave led to only marginal improvements in progression-free survival andoverall survival. Therefore, there is a need in the art to obtain newand improved treatments for such cancers.

SUMMARY OF THE INVENTION

In some aspects, the disclosure provides a pharmaceutical compositioncomprising an effective amount of an Insulin-like Growth Factor 1Receptor antisense oligodeoxynucleotide (IGF-1R AS ODN), whereinadministering the pharmaceutical composition to a subject having M2cells in their circulation, tumor microenvironment, or serum thatpolarizes undifferentiated monocytes towards M2 cells reduces the numberof M2 cells in the subject.

In other aspects, the disclosure provides a method for selectiveelimination of M2 cells in a subject comprising systemicallyadministering to the subject an effective amount of the pharmaceuticalcomposition.

In other embodiments, the disclosure provides a method of treatingcancer by reducing the number of M2 cells comprising systemicallyadministering to a subject suffering from the cancer an effective amountof the pharmaceutical composition.

In yet other embodiments, the disclosure provides a method for enhancingimmune response in a subject comprising systemically administering tothe subject an effective amount of the pharmaceutical composition.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts expression of CD163+ cells in the periphery of patientswith glioma. This subset of monocytes is initiated by the presence ofthe tumor, and this subpopulation supports tumor growth and invasion dueto its angiogenic and immunosuppressive nature. Glioma grade isassociated with the accumulation and activity of cells bearing M2monocyte markers. The presence of this population of M2-like CD163+macrophages within the tumor and similar M2 monocytes in the circulatingperiphery also subverts any pro-inflammatory anti-tumor vaccinestrategies. FIG. 1A. flow cytometry reflecting an increase in CD14+cells in WHO Grade III astrocytomas is depicted. FIG. 1B. graphicalrepresentation comparing levels of CD163+ cells according to WHO grade.Grade III and Grade IV tumors show significantly different % monocytesin PMBC as compared to either normal subjects or WHO Grade IIastrocytomas.

FIG. 2A and FIG. 2B depict uptake of labeled antisense nucleic acidsdirected against Insulin-like Growth Factor 1 Receptor (IFG-1R AS ODN)according to cell type (FIG. 2A) and immunotype (FIG. 2B). Macrophages(CD14+) derived from tumor and matched blood samples in glioma patientsavidly uptake antisense directed against the insulin-like growth factortype 1 receptor (IGF-1R AS ODN).

FIG. 3A and FIG. 3B depict flow cytometry of cells with expression ofInsulin-like Growth Factor 1 Receptor (IFG-1R). Normal peripheralmonocytes polarized to M2 cells in vitro overexpress the IGF-1R comparedto macrophages induced to an M1 polarization. Further, the IGF-1R AS ODNselectively induces cell death in the M2 subpopulation in adose-dependent manner. FIG. 3A details that IGF-1R AS ODN selectivelytargets the removal of M2 macrophages such that in situations wherethese cells predominate, their tumor promoting effects can be eliminatedand therapeutic Th1 immunity can be rescued. Open circles representdifferentiated, unstimulated cells, and closed circles representdifferentiated and stimulated cells. FIG. 3B depicts the difference inmonocyte subset distribution after treatment with IGF-1R AS ODNaccording to macrophage polarization.

FIG. 4 depicts quantification of tumor associated CD163+ cells in apatient throughout the course of treatments. Mean and SD of 5400× fieldswere determined by Aperio quantification. Four time points are provided,a first surgery, first recurrence, second recurrence, and autopsy andthe Aperio CD163+ cells on the y axis. FIG. 4 shows that the methodsdisclosed herein are effective in reducing CD163+ cells for patientsthat have failed standard therapy.

FIG. 5 shows immunohistochemistry for IGF-1R in 6 consecutiveglioblastoma multiforme specimens (FIG. 5A-FIG. 5F). All tumorsdemonstrated IGF-1R immunoreactivity indicating the presence of one ormore IGF-1R-expressing cells in the tumor microenvironment andidentifying IGF-1R as a target for cancer therapy.

FIG. 6A-FIG. 6F depict mass spectrometry of two different sequence lotsof IGF-1R AS ODN. FIG. 6A-FIG. 6C: Avecia lot production of DWAsequence; FIG. 6D-FIG. 6F: Girindus lot production of NOBEL sequence;FIG. 6A and FIG. 6D: stability of AS ODN in lyophilized powder form;FIG. 6B and FIG. 6E: formulation in sterile saline; FIG. 6C and FIG. 6F:formulation in sterile saline. Stability results of IMV118 LOT#GAI-08-060-S3-B1 reveal the smallest degradation product is ˜300 Da,and therefore the measured spectral mass meets the requirement of5709±300 Da and acceptable stability in storage to date from lotrelease. The Avecia sequence (DWA) reveals stability over a nine yearperiod.

FIG. 7 shows that circulating CD68+CD163+ cells are reduced in animalsat least 14 days after receiving one dose of NOBEL (SEQ ID NO: 1)systemically (i.p.) following GL261 implantation in the CNS. NOBEL is an18-mer phosphorothioate oligodeoxynucleotide IGF1-R antisenseoligodeoxynucleotide (AS ODN) starting with six nucleotides downstreamfrom the initiating methionine codon. NOBEL is manufactured by solidphase organic synthesis using well-established methodology in asynthesizer equipped with a closed chemical column reactor usingflow-through technology. Each synthesis cycle sequence on the solidsupport consists of multiple steps, which are carried out sequentiallyuntil the full-length oligonucleotide is established. NOBEL is thenlyophilized, packaged in a HDPE container with screw cap and thenvacuum-heat-sealed inside a 5-mL Mylar pouch for storage at −80° C.Prior to use, the lyophilized powder is dissolved in saline until a 100mg/mL solution is achieved. The resulting solution is sterile filteredthrough a 0.22 μm membrane filter. 1 mL aliquots are filled into USPType 1 glass vials and sealed with an appropriate rubber stopper andaluminum cap prior to storage at −80° C.

For this experiment, white blood cells were stained with biotinylatedanti-mouse CD163 (Biorbyt), washed, and a secondary streptavidin-APCadded. After two washes and fixation, cells were permeabilized andstained intracellularly with anti-mouse CD68-PE, followed by two washeswith perm-buffer and a final PBS wash to close membranes. Samples wererun on a Millipore Guava flow cytometer and analyzed using FlowJo.Samples taken at sacrifice show a substantial shift in WBC populationsfollowing i.p. administration of NOBEL. FIG. 7A. PBS i.p. injectioncontrol; FIG. 7B. NOBEL i.p. injection.

FIG. 8 (FIG. 8A and FIG. 8B) shows that the administration of NOBEL (SEQID NO: 1) alone prior to tumor development is effective at delaying theonset of GL261 cell outgrowth. 20 and 50% of C57 and Tbet knockout mice,respectively, develop tumors after i.p. NOBEL, compared with 60 and 100%of C57 and Tbet knockout animals that received vehicle i.p. (PBS),respectively. Significance was assessed using the log rank test(*=p<0.05).

FIG. 9 shows that Cytosine-phosphorothioate-guanosine-DNA activates TLR9expressed on B-cells and plasmacytoid dendritic cells (DCs).

FIG. 10 shows that antigen-presenting cells take up AS ODN and expressincreased costimulatory molecules and express levels of CD80/83/86 inPBMC before and after AS ODN treatment; mDC, myeloid dendritic cell;pDC, plasmacytoid dendritic cell.

FIG. 11 shows that NOBEL (SEQ ID NO: 1) activates monocyte-deriveddendritic cells (DC) as determined by decreased median fluorescenceintensity. Immature DCs engulfed large amounts of fluorescent proteinresulting in higher fluorescent intensities (depicted by larger bars).Mature DCs (activated) down-regulate endocytosis and as a result, takeup less fluorescent protein and possess low fluorescent intensities(depicted by smaller bars). Treatment of monocyte-derived dendriticcells with IGF-1R AS ODN reveals a striking dose-dependent maturationresponse.

FIG. 12 shows that the CpG motif, 5′G*G motif, and phosphorothioatelinkages all provide an additional maturation stimuli to dendriticcells. FIG. 12A: Immature DCs are highly endocytic and engulf largeamounts of fluorescent protein (upper panel) resulting in higherfluorescent intensities. FIG. 12B: Monocyte-derived DC were incubated inthe presence of various IGF-1R/AS ODN (1 μg/ml) for 24 hrs. LPS-treatedDCs (1 μg/ml) served as a positive control for maturation. Immature DCsengulfed large amounts of fluorescent protein resulting in higherfluorescent intensities (depicted by larger bars). Mature DCs(activated) down-regulate endocytosis and as a result, take up lessfluorescent protein and possess low fluorescent intensities (depicted bysmaller bars). CpG motifs contained in IGF-1R/AS ODN also providematuration stimuli to DCs (see control and LNA DC). Phosphorothioatelinkages provide an additional maturation stimuli to DC (see NOBEL DC).5′ G*G motif provides a third maturational stimuli to DCs (see DWA PTDC). The oligomers tested included SEQ ID NO: 1 (NOBEL), SEQ ID NO: 11(IDT1220 phosphorothioate AS ODN (IDT1220)), SEQ ID NO: 15 (DWAphosphorothioate AS ODN (DWA PT)), SEQ ID NO: 16 (DWA locked nucleicacid AS ODN (LNA)), and SEQ ID NO: 17 (DWA phosphodiester AS ODN (DWAcontrol)).

FIG. 13 shows that FIG. 13A. the DWA sequence at 37° C. maintains ahairpin loop secondary structure (shaded inset) at the 5′ side possiblyaffecting base-pairing to targeted mRNA sequence. FIG. 13B. the NOBEL(SEQ ID NO: 1) sequence at 37° C. has no hairpin loop (shaded insets) atthe 5′ side of a CpG motif for two alternate secondary structures withMP at 18° C., allowing for greater likelihood of targeted base pairingand also CpG.

FIG. 14 shows NOBEL (SEQ ID NO: 1) titration in GL261. Cells were plated20 k per well in 96-well plate with growth media and incubated for 4 hr(37 C, 5% CO₂ humidified); growth media was removed and serum-freeOpti-MEM (100 μL) with desired AS ODN concentration was added to eachwell. Cells were returned to culture for an additional 24 hr. FIG. 14A.Effects of NOBEL titration on IGF-1R expression in GL261 cells. Copynumber IGF-1R versus final mg per well in microtiter plate (or mg per 20k cells). Cells were plated. Significance was determined with ANOVAanalysis (*=P<0.05; **=P<0.001). FIG. 14B. Cells were harvested, stainedwith an antibody specific for mouse IGF-1R, and analyzed with a flowcytometer. Median fluorescence intensity is plotted versus final AS ODNconcentration (mg per 20 k cells). IGF-1R expression was significantlyreduced in GL261 cells treated with 1 mg Nobel AS ODN per well(P<0.001), as well as in cells treated with 0.1 mg Nobel AS ODN per well(P<0.05).

FIG. 15 shows the results of quantitative RT-PCR to assess downstreamdownregulation of hexokinase isotype 2 mRNA. The expression of L13,IGF-1R and HexII genes in NOBEL (SEQ ID NO: 1)-treated cells of thehuman glioma line U118 is linearly correlated. Specific mRNA copynumbers for the housekeeping gene L13 (▾) and hexokinase 2 [HEX] (▪)plotted against IGF-1R copy numbers detected in individual culturestreated with NOBEL at different concentrations are shown. The solid linerepresents the best-fit linear regression line between L13 and IGF-1Rand the dotted line represents the best-fit linear regression linebetween Hex-II and IGF-1R with r² representing the degree of linearity(out of 1.0) and P the significance of the slope.

FIG. 16 shows the cumulative tumor growth in C57/B6 mice injected with10⁶ GL261 cells two weeks post AS ODN treatment. All mice in the AS ODNgroup were injected in the flank once with 10⁶ NOBEL (SEQ ID NO:1)-treated GL261 (overnight AS ODN treatment, 20 mg/5×10⁶ GL261) andchallenged two weeks post treatment in the opposite flank with WT GL261;mice in AS ODN/GL261 mix group were injected in the flank once withNOBEL (20 mg/5×10⁶ GL261) mixed with untreated GL261 cells immediatelyprior to injection and challenged two weeks post treatment in theopposite flank with WT GL261. Tumors developed from the post treatmentchallenge (WT GL261).

FIG. 17 shows that the combination of GL261 cells and NOBEL (SEQ IDNO: 1) at site of administration prevents tumor formation in asubcutaneous model.

FIG. 18 shows that NOBEL (SEQ ID NO: 1) induces radiosensitization.

FIG. 19A-FIG. 19D depicts a safety assessment study. FIG. 19A. overallsurvival of patients in trial; FIG. 19B. overall survival related tointerval between surgeries; FIG. 19C. protocol survival with twosurvival cohorts. Nine patients died of disease progression while onedied of intracerebral hemorrhage and two of sepsis. Overall protocolsurvival was 48.2 weeks and 9.2 weeks, respectively for longer (N=4) andshort (N=8) survival cohorts (log-rank=0.0025). FIG. 19C. Excludingnon-disease progression cause of death, median survival was 48.2 weeksand 10 weeks, respectively for longer (N=4) and short cohorts (N=5)respectively (log-rank=0.0049); FIG. 19D. excluding one outlier (longcohort) linear regression revealed high correlation between protocolsurvival and lymphocyte count at enrollment (R²=0.8, p=0.0028).

FIG. 20A-FIG. 20F depicts radiographic responses of anatomic tumors.FIG. 20A. examples of short survival cohort. TJ12: A-D; TJ10: E-H; A, E:pre-operative T1-gadolinium enhanced axial images; G:T1-gadolinium-enhanced coronal image; C: pre-operative axial FLAIRimage; B, D, F, H: respective 3 month post-operative images; FIG. 20B.examples of longer survival cohort. TJ06: A-D; TJ09: E-H. A, E:pre-operative T1-gadolinium-enhanced axial images; C, F: pre-operativeaxial FLAIR images; B, D, F, H: respective 3 month post-operativeimages; FIG. 20C. relationship between relative cerebral blood volume intumor v. apparent diffusion coefficient in short survival cohort; FIG.20D. longer survival cohort; there is a high correlation between theapparent diffusion coefficient (ADC) and relative cerebral blood volume(rCBV) (R²=0.96, p=0.0005); FIG. 20E. summary of cytokine responses inthe longer survival cohort (N=3); FIG. 20F. example of CD163+ cell lossas it relates to rCBV and ADC over time in patient TJ06; also assay ofactivated nitric oxide synthetase, an agent of hyperemia, reflected asserum nitrate levels (Greiss assay) as they relate to rCBV.

FIG. 21A-FIG. 21C shows an examination of explanted chambers andpathological specimens. FIG. 21A. photomicrograph composite of chamberexplant from TJ09; left column: PBS chamber; right column: vaccinechamber; upper row: H&E stain of outer surface of membranes; lower row:CD163+ immunostain of outer surface membranes; FIG. 21B.immunofluorescent stains for CD163 (red); a. CD163+ TAMs in tumor ofpatient TJ14 at initial resection; b. CD163+ TAMs in tumor of patientTJ14 at recurrence prior to vaccination; CD163+ TAMs were increased; c.CD163+ TAMs in tumor of patient TJ14 at second recurrence; a loss ofTAMs in tumor microenvironment was observed, and CD163 TAMs were foundassociated only with blood vessel; d, e, f. higher magnifications,respectively; FIG. 20C. Aperio immunostain quantification of CD163+ TAMsaccording to stage of treatment (Left Two Panels); also noted aresimilar levels in both Phase I trials but significantly lower levels inundiagnosed, untreated patients who underwent autopsy (Right Panel).

FIG. 22A-FIG. 22E depict serial measurements of immune effector cellshifts and cytokine/chemokine shifts after induction vaccination in thepost-treatment period; longer survival cohort, (patients TJ03, TJ14,TJ06, TJ09); example of short survival cohort, (patient TJ13 for allother short survival cohorts, see FIG. 25). Rows: FIG. 22A. absolute CD4and CD8 counts compared to relative amounts of WBC among PBMC; FIG. 22B.levels of CCL21 and CXCL12;

FIG. 22C. relationship of relative T-cell and macrophage counts; FIG.22D. relationship of relative proportion of CD14+CD16− macrophages withCCR2 and MCP-1 (CCL2); FIG. 22E. putative Th-1 cytokine responses aftervaccinations.

FIG. 23 depicts a summary of cytokine levels (pg/ml) at day 14 for FIG.23A. putative Th-1 cytokines; FIG. 23B. Th-2 associated cytokines, afterPMA/ionomycin stimulation, by survival cohort. Comparison of means(Tukey) and unpaired t-test. Significance at p<0.05. TJ03 was excludedas an outlier with values consistently outside the 95% CI.

FIG. 24 depicts radiographic responses of anatomic tumors. FIG. 24A andFIG. 24C: axial gadolinium-enhanced T-1W images; FIG. 24A and FIG. 24B:patient TJ06 and FIG. 24A and FIG. 24D, patient TJ07; FIG. 24B and FIG.24D: delayed PET/CT images in same axial registration. In FIG. 24B notephotopenia including lack of normal increased metabolism of lefttemporal lobe cortical ribbon compared to right temporal lobe; smallarea of increased metabolism in anterolateral temporal lobe. Themajority of enhancement in FIG. 24A is interpreted as inflammation. InFIG. 24D note distinct correlation of increased metabolism withcorresponding volume of enhancement in FIG. 24C which is interpreted asdisease progression.

FIG. 25 shows a comparison of mean cytokine levels by source (pg/ml)(C-p, PBS chamber; C-v, vaccine chamber; sera; SN, autologous tumor cellsupernatant). CCL21 is significantly elevated in the vaccine chambercompared to both C-p and sera. CCL20 is significantly elevated in C-vand C-p v. sera; CCL19 was significantly elevated in C-v vs. C-p orsera. HSP-70 is significantly elevated over sera; CCL2 is significantlyelevated over sera; CXCL12 is the only cytokine significantly elevatedin sera vs. C-p. *p<0.035, **p<0.025, ***p<0.015, †p<0.004, ††p<0.0002,†††p<0.0001.

FIG. 26A-FIG. 26D depicts an examination of explanted chambers andpathologic specimens. FIG. 26A left panel: Comparison of means fornumber of immunopositive cells/400× field CD163 TAMs at initialdiagnosis v. recurrence prior to vaccination; right panel: matched pairscomparison mean difference 19.2% increase, p<0.0001; FIG. 26B leftpanel: Comparison of means for number of CD163 TAMs at recurrence priorto vaccination v. autopsy; right panel: matched pairs comparison meandifference −26.35% decrease, p<0.0001; FIG. 26C. retrospectivecomparison of CD163 TAMs in paraffin samples from the original trial andthe current trial v. six autopsy specimens from undiagnosed anduntreated glioblastomas; FIG. 26D. assessment of IGF-1R+ cells inparaffin sections obtained at initial diagnosis, recurrence prior tovaccination, and at autopsy.

FIG. 27. FIG. 27A. comparison of means for number of immunopositivecells/400× field detecting CD163 cells by survival cohort; left panel:at diagnosis, long v. short, p<0.0002; right panel: at tumor resectionprior to induction vaccination, long v. short, p<0.0127; FIG. 27B.linear regression of relationship between peripheral and tumorassociated macrophages (R²=0.96, p=0.004).

FIG. 28A-FIG. 28E depicts serial measurements of immune effector cellshifts and cytokine/chemokine shifts after induction vaccination in thepost-treatment period for short survival cohort, (patients TJ01, TJ02,TJ07, TJ08, TJ10, TJ11, and TJ12, respectively); rows: FIG. 28A.absolute CD4 and CD8 counts compared to relative amounts of WBC amongPBMC; FIG. 28B. levels of CCL21 and CXCL12; FIG. 28C. relationship ofrelative T-cell and macrophage counts; FIG. 28D. relationship ofrelative proportion of CD14+CD16− macrophages with CCR2 and MCP-1(CCL2); FIG. 28E. putative Th-1 cytokine responses after vaccinations.

FIG. 29A demonstrates that the vast majority of IGF-1R AS ODN uptakeoccurs with monocytes and neutrophils; FIG. 29B. despite similar uptakeof IGF-1R AS ODN in M1 and M2 cells, increasing concentrations of IGF-1RAS ODN targets selective elimination of M2 CD163+ cells withupregulation of IGF-1R only; FIG. 29C. rate of apoptotic cell death ofCD163+ cells is directly related to the concentration of IGF-1R AS ODN.

FIG. 30A and FIG. 30B depict polarization of monocytes towards M2 cellsby incubation of normal monocytes in cancer patient sera. FIG. 30A.comparison of means for PBS control v. IGF-1R AS ODN (NOBEL, 250 μg)treatment of CD163+ macrophages; FIG. 30B. matched pairs analysisreveals highly significant decrease in M2 cell population.

FIG. 31A and FIG. 31B show that monocytes polarized towards the M2CD163+ phenotype by treatment with sera from patients with differentcancers show upregulation of both CD163 as well as PDL-1; in both casestreatment with AS ODN knocks down both CD163 and PDL-1 by selectivelytargeting this population of cells. FIG. 31A. comparison of means forPBS control v. IGF-1R AS ODN (NOBEL, 250 μg) treatment of CD163+macrophages expressing PDL-1; FIG. 30B. matched pairs analysis revealshighly significant decrease in this cell population reflected assignificant reduction of PDL-1.

FIG. 32 shows that monocytes polarized towards M2 by treatment withIL-10 produce substantially more glutamine (gln) than monocytespolarized towards M1 by treatment with LPS/IFNγ and are therefore morelikely to promote the growth of tumor cells. Normal human monocytes werepolarized towards M1 and M2 in vitro by treatment with LPS/IFNγ and MCSFor IL 10 respectively. FIG. 32A. levels of glutamine accumulating in theculture medium at various time points; FIG. 32B. shows intracellularglutamine levels assessed at 24 hours of culture.

FIG. 33A-FIG. 33C shows the difference in circulating CD163+ monocytesbetween normal individuals and astrocytoma patients. FIG. 33A: normalindividual with ˜6% CD14+ monocytes in their circulation withintermediate levels of CD163. Two changes are observed in the cancerpatient—higher numbers of monocytes and the monocytes have higher levelsof CD163. Other cells (red box) do not have CD163 at all. FIG. 33B:normal individuals can have a wide range of monocytes, due to infectionsetc. (FIG. 33B, cells positive for CD11b+CD14) but these are elevated inpatients with malignant astrocytomas. The histogram in FIG. 33C showsthat monocytes from cancer patients have higher levels of CD163 on theirCD14 monocytes than control cells (red histogram).

FIG. 34 shows tumor-infiltrating M2 monocytes, wildtype IDH1 status, andgadolinium-enhancement by MRI in anaplastic astrocytoma patients definea more aggressive tumor associated with poor prognosis. Formalin-fixed,paraffin-embedded tissues were stained for the IDHR1 mutation R132H(FIG. 34A) and CD163 (FIG. 34B). Representative images for FLAIR (FIG.34C and FIG. 34D, left panels) and gadolinium-enhanced T1-weighted axialMRI (FIG. 34C and FIG. 34D, right panels) are shown for non-enhancing,AIII (IDH1 R132H mutant grade III) (FIG. 34C) and enhancing, AIII-G(IDH1 wild-type grade III with characteristics of glioblastomamultiforme) (FIG. 34D) tumors. Patients were divided into groups basedon these three aforementioned parameters (FIG. 34A-FIG. 34D),specifically, AIII and AIII-G which resemble more aggressive GBM (FIG.34E, FIG. 34F, and FIG. 34G). Results for the presence (R132H⁺) orabsence (R132H⁻) of the IDH1 mutation in 38 randomly selected MRIenhancing and non-enhancing AA patients are shown in FIG. 34E where n.d.represents none detected. The CD163⁺ cell content in excised tumorspecimens was enumerated using an automated cell counting system and ispresented for AA specimens separated by enhancement in FIG. 34F.Box-and-whisker plots indicate the 75^(th), 50^(th), and 25^(th)percentiles while maximum and minimum data values are represented by theupper and lower whiskers. The statistical significance of the differencebetween the groups was assessed by the Mann Whitney test (***, p<0.001).The Kaplan-Meier survival curves of patients segregated based on theaggressiveness of their tumors are presented in FIG. 34G. Statisticallysignificant survival differences between the groups (**) were determinedby the Log-Rank (p=0.0019) and Wilcoxon tests (p=0.0088). The resultsindicate that IDH R132H mutant grade III astrocytomas rarely enhancewith gadolinium and that the accumulation of CD163⁺ M2 cells in tumortissues is associated with the loss of vascular integrity.

FIG. 35 shows that the numbers of circulating monocytes are elevated inAIII and AIII-G patients and express increasing levels of the M2 markerCD163. PBMC from 18 randomly selected Anaplastic Astrocytoma (AA)patients (i.e., patients with astrocytomas characterized morphologicallyby WHO histological criteria as grade III) and 24 normal donors werestained with antibodies specific for CD11b, CD14, and CD163 and assessedby flow cytometry. Forward scatter (FSC) and side scatter (SSC) profileswere used to establish a live cell gate and monocytes were defined aslive cells expressing CD11b and CD14 (FIG. 35A). Representative contourplots for the live gate and analysis of CD11b and CD14 positivity inPBMC from a normal and an AA donor are shown in FIG. 35A where axes arepresented as log scale and the numbers indicate the frequency of gatedcells. FIG. 35B is a summary chart showing the frequency of CD11b⁺CD14⁺monocytes in PBMC from 12 patients with AIII, 6 patients with AIII-G,and 24 normal individuals determined by flow cytometry. The statisticalsignificance of differences in cell percentages between normalindividuals and AA patient subsets was assessed by Student's t test (**,p<0.01). The median fluorescence intensity (MFI) for CD163 staining ofCD11b+CD14+ gated monocytes is overlayed from representative histogramplots of AIII, AIII-G, and normal blood specimens in FIG. 35C. Axes arepresented as log scale. The MFI for CD163-staining of the gated monocytesubset in PBMC samples from the different donor groups are presented inFIG. 35D.

FIG. 36 shows that antibodies present in AIII and AIII-G patient serumthat bind shared antigens on astrocytoma exosomes differ in isotypeprofile. Exosomes, isolated from three astrocytoma patient primary tumorcell lines were coated onto 96-well plates and incubated with patientsera (13 AIII, 8 AIII-G) collected before initial surgery and normalcontrol serum (4). Bound antibodies were detected withfluorescently-conjugated whole IgG (FIG. 36A) or secondary antibodiesspecific for IgG isotypes (FIG. 36B) and the extent of antibody bindingmeasured as MFI.

FIG. 37 shows that soluble factors generally associated with Th1 and Th2immunity are elevated in the sera of AIII and AIII-G patientsrespectively.

FIG. 38 shows that levels of expression in PBMC of genes encoding whiteblood cell phenotypic markers, cytokine and chemokine receptors as wellas their ligands differ between AIII and AIII-G patients. The copynumbers of genes for monocyte phenotypic markers (FIG. 38A),interleukins (FIG. 38B), interleukin receptors (FIG. 38C), CC chemokines(FIG. 38D) and receptors (FIG. 38E), and CXC chemokines (FIG. 38F) andreceptors (FIG. 38G) in PBMC from 17 unselected AA patients wereassessed by high throughput quantitative RT-PCR and normalized to thecopy numbers of the housekeeping gene L13a present in each sample.

FIG. 39 shows that AIII and AIII-G patient subsets can be accuratelydifferentiated by the expression of select immunologically-relevantgenes in PBMC. Discriminant analysis was first used to identify the geneexpression data that best separated AIII and AIII-G patient PBMC (FIG.39A). Principal Component Analysis was then used to determine which ofthese genes, CCL3, CCR4, CCR5, CCR7, CXCL7, IL-15, IL-32, IL-15R,IL-21R, IL-23R, IL-31RA, and CD163, are most effective atdifferentiating the two patient cohorts (FIG. 39B).

DETAILED DESCRIPTION

The present disclosure shows, for the first time, that a criticaldistinction between M1 and M2 cells is that the M2 subpopulationproduces higher levels of the insulin-like growth factor type 1 receptor(IGF-1R) than the M1 subpopulation. This indicates that IGF-1R plays acritical role in the polarization and survival of the M2 cells. Indeed,the present disclosure shows that although both M1 and M2 cells avidlyuptake antisense nucleic acids directed against IGF-1R (IGF-1R AS ODN),the IGF-1R AS ODN induces a selective reduction in M2 cells derived fromcancer patients over M1 cells in a dose-dependent manner. Moreimportantly, the present disclosure shows that the selective reductionof M2 cells leads to a regression of the cancer in these patients.Therefore, this specification provides, for the first time, a viable andefficient mechanism of treating certain cancers by selectively reducingthe number of M2 cells in patients by systemically administering IGF-1RAS ODN.

Additionally, in patients suffering from certain types of cancersincluding, but not limited to, glioma, astrocytoma, breast cancer, headand neck squamous cell cancer, papillary renal cell carcinoma Type II,lung cancer, pancreatic cancer, gall bladder cancer, rectal cancer,classical Hodgkin's lymphoma, ovarian cancer, and colorectal cancer, M2cells cause the tumor environment to be immunomodulatory towards Type 2immunity, which suppresses Type 1 immunity and defeats an immunotherapystrategy. M2 cells, thereby, attenuate induction of therapeuticanti-tumor immunity. Consequently, treatments that seek to improve Th1immunity either fail or have reduced efficacy in view of the M2 cellspresent in these patients. The present disclosure shows for the firsttime that reducing the M2 subpopulation also promotes Type 1 immunity incancer patients. The disclosure shows that by targeting and neutralizingthe M2 cell population, the capacity to engender Type 1, protectiveanti-tumor immunity is restored in cancer patients, thereby facilitatingtreatments using immunotherapy strategies.

Even more significantly, the present disclosure shows that the selectivereduction of M2 cells by administration of the IGF-1R AS ODN provides amechanism for delaying the onset of cancer or even preventing cancer insubjects. Therefore, using the IGF-1R AS ODN to selectively knockdown M2cells provides a new and significant immunotherapy approach for thetreatment and prevention of cancer as well as for enhancing therapeuticimmunity in cancer patients. Therefore, the present disclosure providesnew information about the immune system and supports a therapeuticintervention involving targeted elimination of M2 cells associated withpoor prognosis in patients with a variety of cancers.

Accordingly, the present disclosure provides pharmaceutical compositionscomprising nucleic acids capable of targeting IGF-1R expression in M2cells. The present disclosure also provides methods for the selectivereduction of M2 cells by targeting expression of IGF-1R in these cells.The present disclosure further provides methods for treating cancer bytargeting expression of IGF-1R in M2 cells in patients. Importantly, thepharmaceutical composition of the present invention is effective whenadministered systemically to subjects in need thereof. The ease ofadministration of the pharmaceutical composition facilitates treatmentand enhances patient compliance.

The term “selective as used herein refers to an effect which affects M2cells, but does not affect M1 cells. Alternatively, it may refer to aneffect which affects M2 cells to a greater extent in comparison to M1cells. For example, a selective reduction in the number of M2 cells doesnot affect the number of M1 cells, or causes a greater reduction in thenumber of M2 cells in comparison to a reduction in the number of M1cells.

The term “targeting IGF-1R expression” or “targets IGF-1R expression” asused herein refers to administering a nucleic acid that has a sequencedesigned to bind to the IGF-1R.

The term “M2 cells” as used herein encompasses M2 macrophages presentwithin tumors of a subject and/or M2 monocytes circulating in theperiphery of the subject.

The term “M1 cells” as used herein encompasses M1 macrophages presentwithin the tissues of a subject and/or M1 monocytes circulating in theperiphery of the subject.

As used herein, terms such as “a,” “an,” and “the” include singular andplural referents unless the context clearly demands otherwise.

As used herein, the term “about” when preceding a numerical valueindicates the value plus or minus a range of 10%. For example, “about100” encompasses 90 and 110.

In some embodiments, the disclosure provides a pharmaceuticalcomposition comprising an effective amount of a nucleic acid thattargets IGF-1R expression in M2 cells, wherein administering thepharmaceutical composition to a patient suffering from cancer causes areduction in M2 cells.

In certain embodiments, the disclosure provides a pharmaceuticalcomposition comprising an effective amount of a nucleic acid capable oftargeting IGF-1R expression in M2 cells, wherein administering thepharmaceutical composition to a patient suffering from cancer causes adownregulation of expression of IGF-1R in M2 cells. In otherembodiments, the disclosure provides a pharmaceutical compositioncomprising an effective amount of a nucleic acid capable of targetingIGF-1R expression in M2 cells, wherein administering the pharmaceuticalcomposition to a patient suffering from cancer causes a downregulationof expression of genes other than IGF-1R in M2 cells.

In some embodiments, the nucleic acid downregulates the expression ofgenes downstream of IGF-1R pathway in a cell. In certain aspects, thedownstream gene is hexokinase (Hex II). In some embodiments, the nucleicacid downregulates the expression of housekeeping genes in the cell. Insome aspects, the housekeeping gene is L13.

In some embodiments the nucleic acid is a naturally occurring nucleicacid. In other embodiments, the nucleic acid is a non-naturallyoccurring nucleic acid. In certain aspects the nucleic acid isrecombinantly produced. In some embodiments, the nucleic acid isrecombinantly produced in a microorganism. In some aspects, the nucleicacid is recombinantly produced in bacteria. In other embodiments, thenucleic acid is recombinantly produced in a mammalian cell line. In yetother embodiments, the nucleic acid is recombinantly produced in aninsect cell line.

In certain aspects, the nucleic acid is chemically synthesized. Incertain embodiments, the nucleic acid is manufactured by solid phaseorganic synthesis. In some aspects, the synthesis of the nucleic acid iscarried out in a synthesizer equipped with a closed chemical columnreactor using flow-through technology. In some embodiments, eachsynthesis cycle sequence on the solid support consists of multiplesteps, which are carried out sequentially until the full-length nucleicacid is obtained. In certain embodiments, the nucleic acid is stored ina liquid form. In other embodiments, the nucleic acid is lyophilizedprior to storing. In some aspects, the lyophilized nucleic acid isdissolved in water prior to use. In other embodiments, the lyophilizednucleic acid is dissolved in an organic solvent prior to use. In yetother embodiment, the lyophilized nucleic acid is formulated into apharmaceutical composition. In some aspects the pharmaceuticalcomposition is a liquid pharmaceutical composition. In other aspects,the pharmaceutical composition is a solid pharmaceutical composition.

In some embodiments the nucleic acid is an RNA. In other embodiments,the nucleic acid is a DNA. In yet other embodiments, the nucleic acid isan RNAi molecule. In further embodiments, the nucleic acid is anoligonucleotide.

In certain embodiments, the nucleic acid is an antisense oligomer. Insome aspects, the nucleic acid is an antisense oligodeoxynucleotide (ASODN). Antisense oligomers work at the molecular level by binding to atargeted complimentary sequence of mRNA by Watson and Crick base-pairingrules. The translation of target mRNA is inhibited by an active and/orpassive mechanism when hybridization occurs between the complementaryhelices. In the passive mechanism, hybridization between the mRNA andexogenous nucleotide sequence leads to duplex formation that preventsthe ribosomal complex from reading the message. In the active mechanism,hybridization promotes the binding of RnaseH, which destroys the RNA butleaves the AS ODN intact to hybridize with another complementary mRNAtarget. Either or both mechanisms inhibit translation of a proteincontributing to or sustaining a malignant phenotype. As therapeuticagents, they are far more selective and as a result, more effective andless toxic than conventional drugs. The presence of one or morephosphorothioate modifications stabilize the oligomer by conferringnuclease resistance and thereby increase its half-life.

In some embodiments, the nucleic acid comprises a modified phosphatebackbone. In certain aspects, the phosphate backbone modificationrenders the nucleic acid more resistant to nuclease degradation. Incertain embodiments, the modification is a locked nucleic acidmodification. In other embodiments, the modification is aphosphorothioate linkage. In certain aspects, the nucleic acid containsone or more phosphorothioate linkages. In certain embodiments, thephosphorothioate linkages renders the nucleic acid more resistant tonuclease cleavage. In some embodiments, the nucleic acid may bepartially phosphorothioate-linked. For example, up to about 1%, up toabout 3%, up to about 5%, up to about 10%, up to about 20%, up to about30%, up to about 40%, up to about 50% up to about 60%, up to about 70%,up to about 80%, up to about 90%, up to about 95%, or up to about 99% ofthe nucleic acid may be phosphorothioate-linked. In some embodiments,the nucleic acid is fully phosphorothioate-linked. In other embodiments,phosphorothioate linkages may alternate with phosphodiester linkages. Incertain embodiments, the nucleic acid has at least one terminalphosphorothioate monophosphate.

In some embodiments, the nucleic acid comprises one or more CpG motifs.In other embodiments, the nucleic acid does not comprise a CpG motif. Incertain aspects, the one or more CpG motifs are methylated. In otheraspects, the one or more CpG motifs are unmethylated. In certainembodiments, the one or more unmethylated CpG motifs elicit an innateimmune response when the nucleic acid is administered to a subject. Insome aspects, the innate immune response is mediated by binding of theunmethylated CpG-containing nucleic acid to Toll like Receptors (TLR).In some aspects, the TLR is TLR9. In other aspects, binding of TLR tothe unmethylated CpG-containing nucleic acid causes activation of TLR9.In certain aspects, the activated TLR9 is expressed on B-cells. In otheraspects, the activated TLR is expressed on plasmacytoid dendritic cells.In certain aspects, the activation of TLR9 may be measured by secretionof cytokines by B-cells. In one aspect, the cytokine is IL-6. In anotheraspect, the cytokine is IL-10. In other aspects, the activation of TLR9may be measured by secretion of cytokines by plasmacytoid dendriticcells. In one aspect, the cytokine is IFNα. In another aspect, thecytokine is IFNβ. In yet another aspect, the cytokine is TNFα.

In certain embodiments, the nucleic acid comprises at least one terminalmodification or “cap”. The cap may be a 5′ and/or a 3′-cap structure.The terms “cap” or “end-cap” include chemical modifications at eitherterminus of the oligonucleotide (with respect to terminalribonucleotides), and including modifications at the linkage between thelast two nucleotides on the 5′ end and the last two nucleotides on the3′ end. The cap structure may increase resistance of the nucleic acid toexonucleases without compromising molecular interactions with the targetsequence or cellular machinery. Such modifications may be selected onthe basis of their increased potency in vitro or in vivo. The cap can bepresent at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) orcan be present on both ends. In certain embodiments, the 5′- and/or3′-cap is independently selected from phosphorothioate monophosphate,abasic residue (moiety), phosphorothioate linkage, 4′-thio nucleotide,carbocyclic nucleotide, phosphorodithioate linkage, inverted nucleotideor inverted abasic moiety (2′-3′ or 3′-3′), phosphorodithioatemonophosphate, and methylphosphonate moiety. The phosphorothioate orphosphorodithioate linkage(s), when part of a cap structure, aregenerally positioned between the two terminal nucleotides on the 5′ endand the two terminal nucleotides on the 3′ end.

In certain embodiments, the nucleic acid targets the expression ofspecific genes in a cell. In some embodiments, the nucleic acid targetsthe expression of one or more growth factors in a cell. In someembodiments, the growth factor is Insulin like Growth Factor 1 Receptor(IGF-1R). IGF-1R is a tyrosine kinase cell surface receptor that shares70% homology with the insulin receptor. When activated by its ligands(IGF-I, IGF-II and insulin), it regulates broad cellular functionsincluding proliferation, transformation and cell survival. The IGF-1R isnot an absolute requirement for normal growth, but it is essential forgrowth in anchorage-independent conditions that may occur in malignanttissues. A review of the role of IGF-IR in tumors is provided in Basergaet al., Vitamins and Hormones, 53:65-98 (1997), which is incorporatedherein by reference in its entirety.

In certain embodiments, the nucleic acid is an oligonucleotide directedagainst DNA or RNA of a growth factor or growth factor receptor, suchas, for example, IGF-IR.

In certain embodiments, the cell is a mammalian cell. In otherembodiments, the cell is a cell of the immune system including, but notlimited to, monocytes, neutrophils, eosinophils, basophils, leukocytes,Natural Killer (NK) cells, lymphocytes, T cells, B cells, dendriticcells, mast cells, and macrophages.

In certain embodiments, the cell is a macrophage. In certain aspects,the macrophage is a M2 macrophage. In certain aspects, the M2 macrophageexpresses one or more cell surface markers including, but not limitedto, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, CD206, and/orother M2 macrophage markers commonly known in the art.

In other embodiments, the cell is a monocyte. In certain aspects, themonocyte is a M2 monocyte. In certain aspects, the M2 monocyte expressesone or more cell surface markers including, but not limited to, CD11b,CD14, CD15, CD23, CD64, CD68, CD163, CD204, CD206, and/or other M2monocyte markers commonly known in the art.

In certain embodiments, the nucleic acid downregulates IGF-1R expressionin any cell. In other embodiments, the nucleic acid downregulates IGF-1Rexpression in M2 cells. In certain embodiments, IGF-1R expression in M2cells is downregulated by at least about 1%, at least about 2%, at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, or at least about 95% incomparison to M2 cells not treated with the nucleic acid. IGF-1Rexpression in M2 cells may be measured by quantitative RT-PCR.

In certain aspects, IGF-1R expression in M2 cells is downregulated inabout 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes,about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 6hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hoursafter administration of the nucleic acid to the subject.

In some embodiments, IGF-1R expression in M2 cells remains downregulatedin the subject for at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, or atleast about 6 weeks after receiving one dose of the nucleic acid.

In some aspects, the downregulation of expression of IGF-1R in M2 cellscauses a selective reduction of M2 cells in a subject in comparison toM1 cells. In certain aspects, targeting the expression of IGF-1R in M2cells causes a selective reduction of M2 cells in a subject incomparison to M1 cells.

In certain embodiments, M2 cells in a subject are reduced by at leastabout 2%, at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, or atleast about 95% in comparison to a subject in need of treatment with thenucleic acid targeting IGF-1R expression in M2 cells; for example, thesubject prior to treatment. In some embodiments, the M2 cells in thesubject are reduced by at least about 40%. In other aspects, the M2 cellpopulation is eliminated. For example, after administration of thepharmaceutical composition of the present invention, the M2 cellpopulation may be about 1%, about 2%, about 5%, or about 10% of thepopulation before administration of the pharmaceutical composition. M2cells in a subject may be measured using FACS. In certain aspects, aftertreatment the M2 cells are eliminated; i.e., undetectable by FACS.

In certain aspects, the reduction in M2 cells is observed in about 10minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours,about 12 hours, about 24 hours, at about 48 hours, or about 72 hoursafter administration of the nucleic acid to the patient.

In some embodiments, the reduction in M2 cells in the subject issustained for at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, or atleast about 6 weeks after receiving one dose of the nucleic acid.

In some embodiments, targeting the expression of IGF-1R preventsundifferentiated monocytes from being polarized to M2 cells. In otherembodiments, targeting the expression of IGF-1R in M2 cells causes theM2 cells to either lose their M2 phenotypic and functional properties,or undergo cell death. In certain embodiments, the cell death isnecrosis. In other embodiments, the cell death is apoptosis. Apoptosis,for purposes of this invention, is defined as programmed cell death andincludes, but is not limited to, regression of primary and metastatictumors. Apoptosis is a programmed cell death which is a widespreadphenomenon that plays a crucial role in the myriad of physiological andpathological processes. Necrosis, in contrast, is an accidental celldeath which is the cell's response to a variety of harmful conditionsand toxic substances. In yet other embodiments, targeting the expressionof IGF-1R in M2 cells causes the M2 cells to undergo cell cycle arrest.

In certain embodiments, the nucleic acid of the invention is anantisense deoxynucleotide directed against IGF-1R (IGF-1R AS ODN). Thefull length coding sequence of IGF-1R is shown in SEQ ID NO:19. Incertain aspects, the nucleic acid may have at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 98%, or 100%identity to the IGF-1R AS ODN. Percentage identity can be calculatedusing the alignment program ClustalW2, available atwww.ebi.ac.uk/Tools/msa/clustalw2/using default parameters.

In certain embodiments, the nucleic acid comprises nucleotide sequencescomplementary to the IGF-1R signal sequence, comprising either RNA orDNA. The signal sequence of IGF-1R is a 30 amino acid sequence. In otherembodiments, the nucleic acid comprises nucleotide sequencescomplementary to portions of the IGF-1R signal sequence, comprisingeither RNA or DNA. In some embodiments, the nucleic acid comprisesnucleotide sequences complementary to codons 1-309 of IGF-1R, comprisingeither RNA or DNA. In other embodiments, the nucleic acid comprisesnucleotide sequences complementary to portions of codons 1-309 ofIGF-1R, comprising either RNA or DNA.

In certain embodiments, the nucleic acid is at least about 5nucleotides, at least about 10 nucleotides, at least about 15nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 45nucleotides, or at least about 50 nucleotides in length. In someembodiments, the nucleic acid is from about 15 nucleotides to about 22nucleotides in length. In certain aspects, the nucleic acid is about 18nucleotides in length.

In certain embodiments, the nucleic acid forms a secondary structure at18° C., but does not form a secondary structure at about 37° C. In otherembodiments, the nucleic acid does not form a secondary structure atabout 18° C. or at about 37° C. In yet other embodiments, the nucleicacid does not form a secondary structure at any temperature. In otherembodiments, the nucleic acid does not form a secondary structure at 37°C. In particular embodiments, the secondary structure is a hairpin loopstructure.

In some aspects, the nucleic acid comprises any of SEQ ID NOS: 1-14, orfragments thereof. In certain embodiments, the nucleic acid may have atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 96%, atleast about 98%, or 100% identity to any of SEQ ID NOS: 1-14, orfragments thereof.

In some aspects, the nucleic acid consists of any of SEQ ID NOS: 1-14.In certain aspects, the nucleic acid is SEQ ID NO: 1. SEQ ID NO: 1 isreferred to as NOBEL. NOBEL is an 18-mer oligodeoxynucleotide with aphosphorothioate backbone and a sequence complimentary to codons 2through 7 in the IGF-1R gene. As such, NOBEL, is an antisenseoligonucleotide directed against IGF-1R (IGF-1R AS ODN). The NOBELsequence, derived as the complimentary sequence of the IGF-1R gene atthe 5′ end, is:

5′-TCCTCCGGAGCCAGACTT-3′.

NOBEL has a stable shelf life and is resistant to nuclease degradationdue to its phosphorothioate backbone. Administration of NOBEL can beprovided in any of the standard methods associated with introduction ofoligodeoxynucleotides known to one of ordinary skill in the art.Advantageously, the AS ODNs disclosed herein, including NOBEL, may beadministered with little/no toxicity. Even levels of about 2 g/kg(scaled) based on mice tests (40 μg in the tail vain) did not revealtoxicity issues. NOBEL can be manufactured according to ordinaryprocedures known to one of ordinary skill in the art.

The pharmaceutical compositions disclosed herein contain the nucleicacid in addition to a pharmaceutically acceptable carrier or diluent;for example, the composition may contain saline (0.9% sodium chloride).

Dosages for the nucleic acid in human subjects may be about 0.025 g/kg,about 0.05 g/kg, about 0.1 g/kg, about 0.15 g/kg, or about 0.2 g/kg. Incertain aspects, the nucleic acid is supplied as a lyophilized powderand re-suspended prior to administration. When resuspended theconcentration of the nucleic acid may be about 50 mg/ml, about 100mg/ml, about 200 mg/ml, about 500 mg/ml, about 1000 mg/ml, or a rangebetween those amounts.

In certain embodiments, the subject is an animal. In other aspects, thesubject is a human. In some embodiments, the subject is suffering from adisease. In certain aspects, the disease is cancer. In certainembodiments, the cancer includes, but is not limited to, breast cancer,astrocytoma, head and neck squamous cell cancer, papillary renal cellcarcinoma Type II, lung cancer, pancreatic cancer, gall bladder cancer,rectal cancer, glioma, classical Hodgkin's lymphoma, ovarian cancer, andcolorectal cancer. In certain aspects, the cancer is glioma. Inparticular aspects, the patient has malignant glioma. In particularaspects, the malignant glioma is a recurrent malignant glioma.

In certain embodiments, a subject who is a candidate for treatment withthe nucleic acid is identified by measuring the levels of circulatingmonocytes in their blood. In some embodiments, the candidate has anelevated number of circulating monocytes in comparison to a healthysubject. As used herein, the term “healthy subject” refers to a subjectnot suffering from cancer or any other disease and not in need oftreatment with the nucleic acid of the invention. In some aspects, thecirculating monocytes include, but are not limited to, CD11b+, CD14+,CD15+, CD23+, CD64+, CD68+, CD163+, CD204+, or CD206+ monocytes. Incertain aspects, the levels of one or more circulating monocytes areelevated by at least about 1.3 fold, at least about 1.5 fold, at leastabout 1.8 fold, at least about 2 fold, at least about 3 fold, at leastabout 4 fold, at least about 5 fold, at least about 10 fold, at leastabout 20 fold, at least about 30 fold, at least about 40 fold, at leastabout 50 fold, at least about 60 fold, at least about 70 fold, at leastabout 80 fold, at least about 90 fold, or at least about 100 fold incomparison to a healthy subject. In particular embodiments, the levelsof one or more circulating monocytes are elevated by about 2 fold incomparison to a healthy subject. Levels of circulating monocytes in thesubject may be measured using Fluorescence-Activated Cell Sorting(FACS).

In certain aspects, the subject has an elevated number of circulatingCD14+ monocytes in comparison to a healthy subject. In certain aspects,the levels of circulating CD14+ monocytes are elevated by at least about1.3 fold, at least about 1.5 fold, at least about 1.8 fold, at leastabout 2 fold, at least about 3 fold, at least about 4 fold, at leastabout 5 fold, at least about 10 fold, at least about 20 fold, at leastabout 30 fold, at least about 40 fold, at least about 50 fold, at leastabout 60 fold, at least about 70 fold, at least about 80 fold, at leastabout 90 fold, or at least about 100 fold in comparison to a healthysubject. In particular embodiments, the levels of the circulating CD14+monocytes are elevated by about 2 fold in comparison to a healthysubject.

In certain embodiments, the circulating CD14+ monocytes have an elevatedlevel of CD163 in comparison to a healthy subject. In some aspects, thelevels of CD163 on the circulating CD14+ monocytes are elevated by atleast about 2 fold, at least about 3 fold, at least about 4 fold, atleast about 5 fold, at least about 10 fold, at least about 20 fold, atleast about 30 fold, at least about 40 fold, at least about 50 fold, atleast about 60 fold, at least about 70 fold, at least about 80 fold, atleast about 90 fold, or at least about 100 fold in comparison to ahealthy subject. In particular embodiments, the levels of CD163 on thecirculating CD14+ monocytes are elevated by about 2 fold in comparisonto a healthy subject.

In other embodiments, a subject who is a candidate for treatment withthe nucleic acid has serum that polarizes undifferentiated monocytestowards M2 cells. In certain aspects, incubation of the subject's serawith undifferentiated monocytes induces the expression of one or morecell surface markers on the monocytes including, but not limited to,CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and/or CD206. Inother aspects, incubation of the subject's sera with undifferentiatedmonocytes elevates the expression of one or more cell surface markers onthe monocytes in comparison to monocytes not incubated with thesubject's sera. In certain aspects, the cell surface markers include,but are not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163,CD204, and/or CD206. In some aspects, the levels of one or more surfacemarkers are elevated by at least about 1.3 fold, at least about 1.5fold, at least about 1.8 fold, at least about 2 fold, at least about 3fold, at least about 4 fold, at least about 5 fold, at least about 10fold, at least about 20 fold, at least about 30 fold, at least about 40fold, at least about 50 fold, at least about 60 fold, at least about 70fold, at least about 80 fold, at least about 90 fold, or at least about100 fold in comparison to undifferentiated monocytes not incubated withthe subject's sera. In particular embodiments, the levels of one or moresurface markers are elevated by about 2 fold in comparison toundifferentiated monocytes not incubated with the subject's sera.Monocytes polarized by a subject's sera may be measured using FACS.

In yet other embodiments, a subject who is a candidate for treatmentwith the nucleic acid is identified by performing a tumor biopsy on thesubject. In some embodiments, tumors from the subject are assayed forthe presence of monocytes. In certain aspects, the monocytes include,but are not limited to, CD11b+, CD14+, CD15+, CD23+, CD64+, CD68+,CD163+, CD204+, or CD206+ monocytes. The presence of monocytes in thetumors may be assayed using immunohistochemistry.

In certain embodiments, a subject who is a candidate for treatment withthe nucleic acid shows CD163+M2 cells greater than about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, orabout 50% of the subjects total peripheral blood mononuclear cells(PBMCs). In certain aspects, the subject shows CD163+M2 cells greaterthan about 20% of the subject's total PBMCs.

In certain embodiments, a subject who is a candidate for treatment withthe nucleic acid is suffering from WHO grade II, WHO grade III, or WHOgrade IV tumor. In certain aspects, the subject is suffering from WHOgrade II tumor. In some aspects, the tumor is an astrocytoma. In certainembodiments, the tumor is selected from grade II astrocytoma, AIII (IDH1R132H mutant grade III astrocytoma), AIII-G (IDH1 wild-type grade IIIwith characteristics of glioblastoma multiforme astrocytoma), or gradeIV astrocytoma. In some aspects the grade IV astrocytoma is glioblastomamultiforme.

In some embodiments, a subject who is a candidate for treatment with thenucleic acid is identified by measuring the levels of a specific set ofcytokines. In some embodiments, the subject has elevated levels of thesecytokines in comparison to a healthy subject. In other embodiments, thesubject is identified by detecting specific micro RNA (miRNA) present inthe tumor. In particular embodiments, the subject has elevated levels ofthese miRNAs in comparison to a healthy subject.

In some embodiments, the nucleic acid induces regression of the cancerin the patient. In other embodiments, the nucleic acid induces areduction of the cancer in the patient. In yet other embodiments, thenucleic acid induces an elimination of the cancer in the patient.

In some embodiments, the nucleic acid induces regression of glioma tumorin the patient. In other embodiments, the nucleic acid induces areduction of glioma tumor in the patient. In yet other embodiments, thenucleic acid induces an elimination of glioma tumor in the patient.

In certain embodiments, the nucleic acid is formulated into apharmaceutical composition. In some aspects, the pharmaceuticalcomposition is formulated in a liquid form. In other aspects, thepharmaceutical composition is formulated in a solid form. In certainaspects, the pharmaceutical composition is formulated for oraladministration. In certain embodiments, the pharmaceutical compositionis in the form of a capsule. In other embodiments, the pharmaceuticalcomposition is in the form of a tablet. In some aspects, the tablet is afast-release tablet. In other aspects, the tablet is acontrolled-release tablet. In other aspects, the pharmaceuticalcomposition is formulated for intraperitoneal administration. In yetother aspects, the pharmaceutical composition is formulated forintravenous administration. In further aspects, the pharmaceuticalcomposition is formulated for intramuscular administration.

In certain embodiments, the pharmaceutical composition is introducedinto a diffusion chamber and the diffusion chamber is surgicallyimplanted into the rectus sheath of a subject for a therapeuticallyeffective time (see, for example, U.S. Pat. No. 6,541,036, which isincorporated herein by reference in its entirety).

As discussed, the present disclosure shows that M2 cells (but not M1cells) avidly uptake antisense nucleic acids directed against IGF-1R(IGF-1R AS ODN) and the IGF-1R AS ODN induces a selective reduction inM2 cells derived from cancer patients over M1 cells in a dose-dependentmanner. More importantly, the present disclosure shows that theselective reduction of M2 cells leads to a regression of the cancer inthese patients.

Therefore, in certain embodiments is provided a method for the selectiveelimination of M2 cells in a patient suffering from cancer comprisingadministering to the patient an effective amount of the pharmaceuticalcomposition.

In other embodiments is provided a method of treating cancer bytargeting expression of IGF-1R in M2 cells comprising administering to apatient suffering from the cancer an effective amount of thepharmaceutical composition.

In certain embodiments, the method of treating cancer further comprisescombination therapy. In some embodiments, the combination therapycomprises radiation therapy. In other embodiments, the combinationtherapy comprises chemotherapy. In certain aspects, the radiationtherapy or chemotherapy is administered to the patient subsequent toadministration of the pharmaceutical composition. In certainembodiments, radiation therapy or chemotherapy is administered to thepatient at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 6 hours, at least about 12 hours, at least about24 hours, at least about 48 hours, at least about 72 hours, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days, at least about 12 days, at leastabout 13 days, at least about 14 days, at least about 3 weeks, at leastabout 4 weeks, at least about 5 weeks, or at least about 6 weekssubsequent to administration of the pharmaceutical composition.

In certain aspects, the pharmaceutical composition is administered tothe patient subsequent to administration of the radiation therapy orchemotherapy. In certain embodiments, pharmaceutical composition isadministered to the patient at least about 1 hour, at least about 2hours, at least about 3 hours, at least about 6 hours, at least about 12hours, at least about 24 hours, at least about 48 hours, at least about72 hours, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 3weeks, at least about 4 weeks, at least about 5 weeks, or at least about6 weeks subsequent to administration of the radiation therapy orchemotherapy.

In certain embodiments, the radiation therapy includes, but is notlimited to, internal source radiation therapy, external beam radiationtherapy, and systemic radioisotope radiation therapy. In certainaspects, the radiation therapy is external beam radiation therapy. Insome embodiments, the external beam radiation therapy includes, but isnot limited to, gamma radiation therapy, X-ray therapy, intensitymodulated radiation therapy (IMRT), and image-guided radiation therapy(IGRT). In certain embodiments, the external beam radiation therapy isgamma radiation therapy.

In certain embodiments, the AS ODN may be administered pre-operatively;for example prior to surgery to reduce tumor burden. For example, the ASODN may be administered up to 24 hours, up to 36 hours, up to 48 hoursor up to 72 hours before surgery. In particular aspects, thepharmaceutical composition may be administered about 48 to about 72hours before surgery. Typically, in such circumstances, theadministration is by intravenous bolus.

As discussed, in patients suffering from certain cancers including, butnot limited to, glioma, astrocytoma, breast cancer, head and necksquamous cell cancer, papillary renal cell carcinoma Type II, lungcancer, pancreatic cancer, gall bladder cancer, rectal cancer, classicalHodgkin's lymphoma, ovarian cancer, and colorectal cancer, M2 cellscause the tumor environment to be immunomodulatory towards Type 2immunity, which suppresses Type 1 immunity. M2 cells, thereby, attenuateinduction of therapeutic anti-tumor immunity. Table 1 below, forexample, summarizes possible immune modulations attributed to gliomasthat may be at least in part caused by M2 cells in gliomas.

TABLE 1 Immunomodulatory Capabilities of Gliomas. ModulationsConsequence Systemic and regional depletion of type 1 Lack ofantigen-specific recognition of tumor cells Th cells by the Th1 cellsthat drive CTL and NK responses Lack of MHC Class I molecules on tumorLoss of recognition by cytotoxic T cells cells MHC class II expressionon tumor cells Regional depletion of Th1 cells TGFβ production by tumorcells Suppression of certain T-, B- and NK-cell and macrophagesresponses Prostaglandin E₂ and IL-10 Suppression of Th1 and professionalAPC production by tumor cells functions Production of colony-stimulatingfactors Recruitment and polarization of macrophages Chemokine productionby tumor cells Chemotaxis of T cells and macrophages into tumor tissue;differentiation of macrophages Presence of IL-1 autocrine loop in tumorPartial activation of T cells and macrophages cells Production of IL-1RAby tumor cells Regulation of IL-1 autocrine loop; suppression of IL-1mediated immune cascade reaction Lack of IFN-α/β genes in tumor cellsReduced innate immune reactivity M2, MDSC and Treg infiltration ingliomas Suppression of anti-tumor cytolytic T cell response

Treatment of such cancer patients with the nucleic acid of the inventionwould eliminate or modify the M2 cells, which would have directinhibitory consequences for tumor growth as well as and promote immunityresponse. Furthermore, it would be preferential in certain embodimentsto treat such patients with a combination of the nucleic acid and avaccination to promote immunity. Accordingly, in certain embodiments, itis advantageous to provide a method of treatment, wherein the nucleicacid is provided, alone, or in combination with a further medicament,for selectively targeting M2 cells. Through the elimination of thesecells, tumor production and promotion is mitigated and reduced, andimmune modulating factors are further modified.

Therefore, in certain embodiments is provided a method for enhancingimmune response in a patient suffering from cancer by targetingexpression of IGF-1R in M2 cells comprising administering to a patientsuffering from the cancer an effective amount of the pharmaceuticalcomposition.

Combination Therapy

The reduction in M2 cells may be accomplished along with stimulation ofan anti-tumor immune response, referred to herein as a vaccinationtherapy. In certain embodiments, the vaccination therapy comprisesplacing tumor cells cultured in vitro or ex vivo in a mediumsupplemented with a pro-apoptotic agent for a period of time, such as,for example, 3 to 48 hours, washing the tumor cells with buffer toeliminate any trace of the pro-apoptotic agent, and subsequentlytransferring the cells to a diffusion chamber, which is then implantedinto the subject (see, for example, U.S. Pat. No. 6,541,036, which isincorporated herein by reference in its entirety). In certainembodiments, the diffusion chamber contains tumor cells which arederived from the same type of tumor to which regression is induced bythe pharmaceutical composition of the present invention. In otherembodiments, the tumor cells placed in the diffusion chamber are of adifferent type than the tumor to which regression is induced.

In certain embodiments, the diffusion chamber is implanted into asubject prior to systemically administering the pharmaceuticalcomposition of the present invention to the subject. In otherembodiments, the diffusion chamber is implanted into a subjectsubsequent to systemically administering the pharmaceutical composition.In yet other embodiments, the diffusion chamber implanted into a subjectalso contains the pharmaceutical composition. In some aspects, adiffusion chamber containing the pharmaceutical composition andautologous tumor cells, treated in vitro or ex vivo as described above,is implanted into a subject. In other aspects, a diffusion chambercontaining the pharmaceutical composition and tumor cells from asubject, treated in vitro or ex vivo as described above, is implanted tothe subject. In certain embodiments, implanting the diffusion chamberinto a subject further reduces the number of M2 cells in the subject incomparison to a subject who is administered the pharmaceuticalcomposition alone.

In some embodiments, the chamber is implanted into the subject'sabdomen. In certain embodiments, the diffusion chamber is surgicallyimplanted into the rectus sheath of a subject for a therapeuticallyeffective time.

In some aspects, the pharmaceutical composition is administered to thesubject subsequent to the administration of the vaccination therapy. Incertain aspects, the pharmaceutical composition is administered to thesubject at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 6 hours, at least about 12 hours, at least about24 hours, at least about 48 hours, at least about 72 hours, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days, at least about 12 days, at leastabout 13 days, at least about 14 days, at least about 3 weeks, at leastabout 4 weeks, at least about 5 weeks, or at least about 6 weekssubsequent to administration of the pharmaceutical composition. Incertain embodiments, the vaccination therapy is administered to thesubject at least about 48 hours subsequent to the administration of thevaccination therapy.

In certain aspects, the vaccination therapy is administered to thesubject subsequent to the administration of the pharmaceuticalcomposition. In certain embodiments, the vaccination therapy isadministered to the subject at least about 1 hour, at least about 2hours, at least about 3 hours, at least about 6 hours, at least about 12hours, at least about 24 hours, at least about 48 hours, at least about72 hours, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 3weeks, at least about 4 weeks, at least about 5 weeks, or at least about6 weeks subsequent to administration of the pharmaceutical composition.In certain embodiments, the vaccination therapy is administered to thesubject at least about 48 hours subsequent to the administration of thepharmaceutical composition.

In some embodiments, the method for enhancing immune response comprisesadministering a second pharmaceutical composition subsequent to thevaccination therapy. In certain embodiments, the second pharmaceuticalcomposition is administered to the subject at least about 1 hour, atleast about 2 hours, at least about 3 hours, at least about 6 hours, atleast about 12 hours, at least about 24 hours, at least about 48 hours,at least about 72 hours, at least about 4 days, at least about 5 days,at least about 6 days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, at least about 14 days, atleast about 3 weeks, at least about 4 weeks, at least about 5 weeks, orat least about 6 weeks subsequent to administration of the vaccinationtherapy.

In other embodiments, the vaccination therapy is administered to thesubject subsequent to the administration of the second pharmaceuticalcomposition. In certain embodiments, the vaccination therapy isadministered to the subject at least about 1 hour, at least about 2hours, at least about 3 hours, at least about 6 hours, at least about 12hours, at least about 24 hours, at least about 48 hours, at least about72 hours, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9days, at least about 10 days, at least about 11 days, at least about 12days, at least about 13 days, at least about 14 days, at least about 3weeks, at least about 4 weeks, at least about 5 weeks, or at least about6 weeks subsequent to administration of the second pharmaceuticalcomposition.

In certain embodiments, the pharmaceutical composition and the secondpharmaceutical composition are the same. In other embodiments, thepharmaceutical composition and the second pharmaceutical composition aredifferent.

Typically, the tumor cells are irradiated prior to implantation; forexample, the cells may be treated with gamma irradiation at an amount ofabout 1 Gy, about 2 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 10 Gy,or up to 15 Gy. In certain embodiments, the cells may be irradiated atleast once, at least twice, at least three times, at least four times,or at least five times.

In certain embodiments is provided a method for preventing or delayingcancer in a subject by targeting expression of IGF-1R in M2 cellscomprising administering to with the subject an effective amount of thepharmaceutical composition. In some embodiments, the subject is ananimal. In other embodiments, the subject is a human. In certainaspects, the human is predisposed to the cancer by virtue of beingcontinuously exposed to one or more carcinogens that increase the riskof that cancer. In certain aspects, these carcinogens include, but arenot limited to cigarette smoke, tobacco, and asbestos. In other aspects,the human is genetically predisposed to the cancer.

In certain embodiments, are provided methods for treating, preventing,or delaying diseases including, but not limited to, Alzheimer's disease,inflammatory bowel disease, insulin resistance in type 2 diabetes, andpsoriasis in a subject by targeting expression of IGF-1R in M2 cellscomprising administering to with the subject an effective amount of thepharmaceutical composition.

EXAMPLES Example 1: Immunohistochemistry for IGF-1R in GlioblastomaMultiforme Specimens

Original magnification 200× (panels A, B, C, D), 400× (panel E). Inorder to evaluate the relevance of IGF-1R expression in glioblastomamultiforme, 18 consecutive glioblastoma cases were stained withanti-IGF-1R alpha (Santa Cruz Biotechnology, Santa Cruz, Calif.).immunohistochemistry for IGF-1R alpha was performed on routine formalinfixed paraffin embedded sections (FIG. 5). Steam heat-induced epitoperetrieval of the sections enhanced with Target Retrieval Solution(#1699, Dako Corporation, Carpinteria, Calif.) was followed by automatedimmunostaining (Dako autostainer, model #LV-1, Dako Corporation) orIGF-1R alpha (Santa Cruz Biotechnology, Santa Cruz, Calif.) at adilution of 1:500. Detection as achieved with a rabbit secondaryantibody MACH 3 Rabbit HRP Kit, Biocare Medical) and AB solution(3,3′-diaminobenzidine in chromagen solution, Dako Corporation). Alltumors demonstrated IGF-1R immunoreactivity.

Example 2: Drug Substance, Formulation and Stability of NOBEL

As described above, NOBEL is an 18-mer oligodeoxynucleotide has aphosphorothioate backbone and is as an antisense directed against theinsulin-like growth factor type 1 receptor (IGF-1R AS ODN) starting withsix nucleotides downstream from the initiating methionine codon. Themolecular weight for the free acid is 5708.71 Daltons. The molecularweight for the sodium salt is 6082.40 Daltons. The sequence of NOBEL,derived as the complimentary sequence of the IGF-1R gene at the 5′ end,is:

5′-TCCTCCGGAGCCAGACTT-3′.

NOBEL is manufactured by solid phase organic synthesis usingwell-established methodology in a synthesizer equipped with a closedchemical column reactor using flow-through technology. Each synthesiscycle sequence on the solid support consists of multiple steps, whichare carried out sequentially until the full-length oligonucleotide isestablished. The drug substance, which is a lyophilized powder, ispackaged in a HDPE container with screw cap and then vacuum-heat-sealedinside a 5-mL Mylar pouch for storage at −80° C.

The drug product consists of the new drug substance dissolved in salineuntil a 100 mg/mL solution is achieved. The resulting solution issterile filtered through a 0.22 μm membrane filter. 1 mL aliquots arefilled into USP Type 1 glass vials and sealed with an appropriate rubberstopper and aluminum cap prior to storage at −80° C. This formulationhas proven to be stable over nine years (last test date) in twoindependent formulations when reconstituted in sterile normal saline(FIG. 6).

Example 3: Preclinical Assessment of Toxicity in Mice

The purpose of this study was to evaluate the toxicity of the testarticle, NOBEL, when administered as a single dose via intravenousinjection to mice; after dosing, animals were observed postdose forapproximately 48 hours (Day 3 interim sacrifice) or 14 days (Day 15terminal sacrifice) to assess the reversibility, persistence, or delayedoccurrence of effects. This study was conducted according to GoodLaboratory Practice and compliant with standards established by the Foodand Drug Administration. Male and female Crl:CD1(ICR) mice were assignedto groups, and doses were administered as indicated in the followingtable. Animals were dosed once at a volume of 100 μL via intravenousinjection in a tail vein with vehicle/diluent [0.9% Sodium Chloride forInjection, USP (sterile saline)] or NOBEL (provided as 100 mg/mL insterile saline) (see Table 2).

TABLE 2 NOBEL Dosing Regimen in Mice No. of Animals^(b) Dose Level DoseConcentration Group^(a) Male Female (mg/mouse) (mg/mL) 1 (Control) 15 150 0 2 (Low) 15 15 0.01 0.01 3 (High) 15 15 0.04 0.04 ^(a)Group 1received vehicle/diluent (sterile saline) only. ^(b)Animals designatedfor interim sacrifice (10 animals/sex/group) were sacrificedapproximately 48 hours after dose administration. Animals designated forterminal sacrifice (five animals/sex/group) were sacrificed 14 daysafter dose administration.

Assessment of toxicity was based on mortality, clinical observations,body weight, food consumption, and clinical and anatomic pathology. Allanimals survived to their scheduled necropsy on Day 3 or 15 of thedosing phase. No test article-related clinical observations or changesin food consumption occurred. No statistically significant effects onbody weight were observed. However, slightly lower body weight (93.8% ofcontrols) occurred by Day 15 of the dosing phase in males given 0.04mg/mouse. This trend was observed as early as Day 8 of the dosing phasein these males and was considered test article-related but not adversebecause no clinical changes in body condition or hydration statusoccurred. No test article-related body weight effects were observed inmales given 0.01 mg/mouse or in females.

NOBEL administration had no effect on clinical pathology test results.Among interim sacrifice animals, lung weights in females given 0.01mg/mouse, testis weights in males given 0.01 or 0.04 mg/mouse, andthymic weights in males given 0.04 mg/mouse were higher. Among terminalsacrifice animals, brain weights in males given 0.01 or 0.04 mg/mouseand lung weights in females given 0.04 mg/mouse were higher. None ofthese organ weight changes had any correlative microscopic observations.Test article dependency was unknown. Regardless, none of the weightdifferences could be considered adverse. None of the macroscopic ormicroscopic observations were considered test article-related.

In conclusion, NOBEL given as a single intravenous bolus injection tomale and female mice at a dose level of 0, 0.01, or 0.04 mg/mouse waswell tolerated and not associated with any clinical observations,changes in food consumption, clinical pathology changes, or macroscopicor microscopic changes. Minor, test article-related changes in bodyweight and increases in brain and lung weights of uncertain relationshipto the test article were not considered adverse. Therefore, the noobserved adverse effect level is considered to be 0.04 mg/mouse.

Example 4: Selective Knockdown of M2 (CD163+) Macrophages

NOBEL selectively knocks down CD163+ cells in both humans and mice.Serum derived from patients with malignant gliomas as well as a varietyof other cancers including, but not limited to, astrocytoma, breastcancer, head and neck squamous cell cancer, papillary renal cellcarcinoma Type II, lung cancer, pancreatic cancer, gall bladder cancer,rectal cancer, classical Hodgkin's lymphoma, ovarian cancer, andcolorectal cancer will differentiate monocytes into the CD163+ phenotypethat take up NOBEL at low micromolar concentrations resulting inknockdown of this phenotype.

In a C57B/6 mouse model intraperitoneal antisense administration twentydays after flank tumor inoculation resulted in knockdown oftumor-induced CD163 cells for at least 14 days (see FIG. 7). GL261 cellswere implanted in the flanks of C57BL/6 and Tbet−/− mice, and then atapproximately 20 days later animals received either PBS or 4 mg NOBELalone as a systemic, intraperitoneal injection (FIG. 8). Tbet−/− micethat are unable to mount anti-tumor type 1 immunity and reject GL261tumors were included to differentiate between effects on the balance oftype 1 and type 2 immunity versus knockdown of M2 cells. In both cases,the administration of a single dose of NOBEL alone just prior to thedetection of palpable tumors delayed the formation of tumors forsignificant periods. Long term survival was also promoted with 80% ofC57 mice and 50% of Tbet knockout mice failing to grow tumors (FIG. 8).Mixing NOBEL with GL261 cells has little effect on tumor growth in theabsence of an intact immune system (Morin-Brureau et al, Cancer Immunol.Immunother., 64:447-457 (2015)) and a mix of NOBEL with GL261 cells atimplantation in Tbet mice also does not interfere with tumor growth. Theimportant conclusion here is that the effects of IGF-R1 AS ODN on GL261at the induction of tumor immunity are distinct from those acting upontumor growth some time later. One requires the co-administration oftumor as antigen and NOBEL as immune stimulant while the other hassystemic effects that are independent of the response to antigen. Theknockdown of M2 cells and possibly other cells involved in promotingtumor growth is evidently sufficient to prevent tumor growth even whenthe recipient animal is unable to mount a therapeutic type 1 response.The data suggests that the loss of M2 cells from the tumormicroenvironment results in failure of the tumor cells to thrive.

Example 5: Administration of IGR-1R AS ODN to Glioma Patients

From the current studies we determined suitable doses to administer inrecurrent glioma cohorts glioma in the following dosing schedules: 0.025g/kg, 0.05 g/kg, 0.1 g/kg, 0.15 g/kg, and 0.2 g/kg.

Patients receiving one of the above doses are patients having recurrentglioma and who receive escalating pre-operative intravenous bolusinfusion of the IGF-1R 48 to 72 hours before surgery. A second bolusinfusion may be further administered depending on quantitation of the M2cell population. This dosing schedule also works for other cancersincluding, but not limited to, astrocytoma, breast cancer, head and necksquamous cell cancer, papillary renal cell carcinoma Type II, lungcancer, pancreatic cancer, gall bladder cancer, rectal cancer, classicalHodgkin's lymphoma, ovarian cancer, and colorectal cancer

Example 6: Different IGF-1R AS ODN Sequences

Different IGF-1R antisense sequences are bioactive in some or all of themulti-modality effects of the NOBEL sequence (5′-TCCTCCGGAGCCAGACTT-3′(SEQ ID NO: 1)). The 18-mer NOBEL sequence has both IGF-1R receptordownregulation activity as well as TLR agonist activity, and furtherexperimentation in mice suggest that both activities are necessary forin vivo anti-tumor immune activity. While the AS ODN molecule hasanti-tumor activity, the complimentary sense sequence does not, despitealso having a CpG motif. The entire open reading frame of the IGF-1Rexon (4104 base pairs) was surveyed and ten additional CpG motifs,including IDT1220, were identified, (SEQ ID NOs: 2-11) (Table 3). Inaddition, several additional IGF-1R antisense sequences (SEQ ID Nos:12-14) that did not contain CpG motifs were also identified (Table 3).

TABLE 3 Potential additional downstream sequences forIGF-1R AS ODN Formulation# Corresponds SEQ to IGF-1R IDSequences with ACGA Motif Codons NO: 5′-TCCTCCGGAGCCAGACTT-3′ 2-7 15′-TTCTCCACTCGTCGGCC-3′ 26-32 2 5′-ACAGGCCGTGTCGTTGTC-3′ 242-248 35′-GCACTCGCCGTCGTGGAT-3′ 297-303 4 5′-CGGATATGGTCGTTCTCC-3′ 589-595 55′-TCTCAGCCTCGTGGTTGC-3′ 806-812 6 5′-TTGCGGCCTCGTTCACTG-3′ 1,033-1,0397 5′-AAGCTTCGTTGAGAAACT-3′ 1,042-1,048 8 5′-GGACTTGCTCGTTGGACA-3′1,215-1,221 9 5′-GGCTGTCTCTCGTCGAAG-3′ 1,339-1,345 105′-CAGATTTCTCCACTCGTCGG-3′ 27-34 11 5′-CCGGAGCCAGACTTCAT-3′ 1-6 125′-CTGCTCCTCCTCTAGGATGA-3′ 407-413 13 5′-CCCTCCTCCGGAGCC-3′ 4-8 14

Example 7: Activation of Toll-Like Receptor 9 by NOBEL

The front-line defense in the immunologic response to invading pathogensinvolves interactions between pathogen structures and an array ofreceptors including toll-like receptors (TLRs) that activate the innateimmune system. This arm of the immune system recognizes generic classesof molecules produced by a variety of pathogens including bacteria,viruses, fungi, and parasites, all of which are essential for thesurvival of the invading pathogen. These pathogen-associated molecularpatterns, or PAMPS, are recognized by pathogen related receptors, orPRRs, expressed in immune effector cells, notably dendritic cells. ThesePRRs were named toll-like receptors due to homology to the toll proteincharacterized in drosophila originally characterized by Nusslein-Volhardin 1991. Stein et al., Cell 65:725-35 (1991). The activating effects ofTLRs, are important in directing the type of ensuing adaptive immuneresponse. There are currently 10 known human TLRs and TLR9 is ofparticular interest. TLR9 binds fragments of DNA that include anunmethylated cytosine-guanosine sequence unique to bacteria and viruses.The human CpG dinucleotide is invariably methylated, renderingautologous human DNA tolerogenic if exposed to the immune system. Theunmethylated CpG motif, particularly when nested in a favorable flankingnucleotide hexamer sequence, elicits a powerful innate immune response(FIG. 9). When compared to antisense gene translation silencing druglevels, responses are seen at 1000-fold lower concentrations.

The NOBEL sequence was found to activate both plasmacytoid DCs and Bcells. In vitro AS ODN uptake experiments with PBMC were performed andassayed for activation of immune cell subsets. The highest uptake of ASODN occurred in endocytic antigen presenting cells: monocytes, dendriticcells (DC), and B cells while negligible uptake was observed in T cellsor NK cells. AS ODN-treated plasmacytoid dendritic cells (pDC), and Bcells, increased expression of costimulatory molecules important in Tcell activation (CD80, 83, and 86). Despite observing the highest levelsof AS ODN uptake, expression levels of CD80, 83, and 86 were unalteredin monocytes and myeloid dendritic cells (FIG. 10).

Example 8: Dendritic Cell Activation and Maturation after NOBELTreatment

Treatment of monocyte-derived dendritic cells with NOBEL reveals astriking dose-dependent maturation response (FIG. 11). Immature DC wereobtained by culturing CD14+ PBMC in rGM-CSF and rIL-4 for 4 days.Immature DC were treated with NOBEL for 36 hours. Poly I:C was used as apositive control for DC maturation. Treated-DC were harvested, incubatedwith a fluorescent protein, and analyzed with a flow cytometer. Highendocytic capacity is a hallmark of immature DC which is rapidly anddramatically reduced upon maturation signals. NOBEL treatment decreasedendocytic capacity in DC in a dose-dependent manner.

Example 9: Optimal AS ODN Sequence

As a guideline to screening more IGF-1R AS ODN sequences, sequences atthe 5′ end of a targeted mRNA transcript have the greatest likelihood ofbinding to an mRNA sequence according to Watson-Crick base-pairingrules. This biological activity is attributable to the favorablesequence which yields a higher probability of a linear molecule at bodytemperature unlike the DWA sequence which is more likely to form astable 5′ hairpin loop at body temperature (FIG. 13). As noted above,the biological activity is attributable to include accessibility to thetargeted mRNA sequence as well as the unmethylated CpG dinucleotidewhich should be accessible from the 5′ end of the molecule and ideally alinear molecule at body temperature. Despite these guidelines the DWAsequence, while having no capability of IGF-1R downregulation, appearedbetter at DC maturation (FIG. 12). The biological activities of thedifferent IGF-1R AS ODNs are summarized in Table 4 below.

TABLE 4 Summary of Biological Activities of Different IGF-1R AS ODNs. M2IGF-1R In vivo IGF- CpG/APC Macrophage Down- Vaccine Radio- SEQ.Formulation 1R activation inhibition regulation capability sensitizerNobel Phosphorothioate 2-7 + + + + + DWA Phosphorothioate 4-9 ++ + − +N/A DWA Locked nucleic acid 4-9 N/A N/A N/A N/A N/A IDT1220Phosphorothioate 407-413 +++ + − N/A N/A Avanti-10 p-ethoxy in neutral2-7 N/A + N/A N/A N/A (BioPath lipid carrier Holdings)

Example 10: Downregulation of IGF-1R: Summary of Biospecificity andBioactivity of NOBEL

Two assays were designed to assess the biospecificity of the AS ODNsequence:

a. Quantitative RT-PCR to assess downregulation of IGF-1R mRNA—thisassay was designed to confirm Watson-Crick base-pairing between cellularIGF-1R transcribed mRNA and the AS ODN. GL261 mouse cells were obtainedfrom NCI-Frederick DTP, DCTD Tumor Bank Repository (Frederick, Md.) andin RPMI supplemented with 10% FBS, 4 Mm L-glutamine (Fisher), 50 μg/mLgentamicin (GIBCO) and 0.05 Mm 2-ME (Sigma). As shown in FIG. 14, IGF-1Rexpression was significantly reduced in GL261 cells treated with 1 mgNobel AS-ODN per well (P<0.001), as well as in cells treated with 0.1 mgNobel AS ODN per well (P<0.05).

b. Quantitative RT-PCR to assess downstream downregulation of hexokinaseisotype 2 mRNA—IGF-1 induces hexokinase RNA expression in cancer cells.It was predicted that reduced IGF-1R activation by IGF-1 as aconsequence of IGF-1R downregulation by AS-ODN treatment should lead toa similar reduction in hexokinase expression. As shown in FIG. 15, thisis the case with the reduction in IGF-1R mRNA being highly correlatedwith downregulation of hexokinase II. A 90% reduction in IGF-1R copynumber corresponded to a 90% reduction in hexokinase II copy number. Adownregulation of housekeeping genes was also expected, in this caseseen as a 75% decrease in L13, since inhibition of the IGF-1R slowsgrowth kinetics and metabolism in vitro.

Example 11: Bioactivity of the IGF-1R AS ODN: Mouse Flank Model toAssess Vaccine Capability of the NOBEL Sequence Against Tumor Challenge

C57/BL6 mice were obtained from Jackson Laboratory (Bar Harbor, Me.) andTaconic Farms, Inc. and used between 8 and 10 weeks of age. Mice wereanesthetized in a chamber containing isoflurane and injected in theflank with 10⁶ GL261 in 100 μL PBS using a 1 mL BD Falcon syringe and21G BD needle (Fisher). AS ODN GL261 cell preparations were injected inthe left flank whereas wild-type GL261 cells were injected in the rightflank two weeks later. Mice were checked at least twice a week for tumordevelopment. It should be noted for these studies that GL261 cells arewell known to be immunostimulatory when placed in the flanks of congenicmice such that roughly 50% of such animals are expected to developanti-tumor cell immunity in the absence of intervention. As seen in FIG.16, pretreatment with AS-ODN treated GL261 cells reduced WT GL261 tumorgrowth from 53% in control mice to 13%, whereas pretreatment with aGL261 AS ODN mixture (4 mg NOBEL per 10⁶ GL261) reduced WT GL261 tumortake to 0%. Of interest, the efficacy of the vaccine was lost when GL261and NOBEL were injected in opposite flanks of the mouse (FIG. 17). Thesedata suggest that although AS ODN-treated GL261 and the antisensemolecule contribute to an anti-tumor response, the most effectivevaccine involves simultaneous injection of autologous tumor cells withthe IGF-1R AS ODN. Also of interest, the NOBEL sense sequence, which ispalindromic around the CpG motif, is not effective at stimulation ofanti-tumor immunity suggesting that the biological effectiveness of theCpG motif is related to the bioactivity of the IGF-1R AS ODN beyond theCpG motif alone.

Example 12: NOBEL is Capable of Radiosensitization

U118 cells were incubated in the presence or absence of NOBEL (4 mg/10⁷cells) for 24 hrs. Cells were harvested, irradiated, and returned toculture (with or without new NOBEL) in the presence of Click-iTedureagent (10 μM final concentration). Following a 72 h incubation,cultures were developed according to the manufacturer's protocol. Asshown in FIG. 18, Nobel AS ODN caused radiosensitivity of U118 humanglioblastoma cells throughout a range of radiation doses from one tofifteen Gy with an isoeffect plateau beyond 5 Gy.

Example 13: Treatment of Astrocytoma

WHO Grade IV astrocytoma (glioblastoma) is a uniformly fatal primaryintracranial malignancy with a median survival of 18 months. Twelvepatients diagnosed with recurrent glioblastoma who were judged to begood surgical candidates were enrolled for treatment. All patients hadfailed standard therapy including surgery, temozolamide chemotherapy andconformal radiation therapy. A summary of enrolled patients and theirdisease courses is included in Table 5. All patients were treated with a3 month course of subcutaneous enoxaparin at 40 mg/day.

TABLE 5 Interval Original Lymphocyte between # lymphocyte count atsurgeries chambers count enrollment Previous IDH-1 Subject Age KPS(weeks) implanted (cells/mm2) (cells/mm2) treatments mutation TJ01 39 70177 10  N/A 400 S, RT + TMZ, − Bev TJ02 57 80 90 9 N/A 1570 S, RT + TMZ− TJ03 75 70 32 7  700 300 S, RT + TMZ − TJ06/R¹ 66 80 54 8 2000 1300 S,RT + TMZ − TJ07 43 80 215 10   500 430 S, RT + TMZ + TJ08 55 80 52 81000 500 S, RT + TMZ − TJ09 57 80 61 7 1400 300 S, RT + TMZ − TJ10 47 60376 7 N/A 1800 S, RT + TMZ − TJ11 39 70 32 11* 2400 200 S, RT + TMZ −TJ12 60 80 74 7 1100 600 S, RT + TMZ − TJ13 64 80 182 11  N/A 2100 S,RT + TMZ − TJ14/R 77 90 30 9/11 1800 1100 S, RT + TMZ − ¹Compassionateretreatment; *Protocol amendment to include control chamber filled withphosphate buffered saline; S: surgery; RT: radiation therapy; TMZ:temozolamide chemotherapy; Bev: bevacizumab chemotherapy; IDH-1:isocitrate dehydrogenase-1

The combination product consisting of autologous tumor cells removed atsurgery then treated overnight with an IGF-1R As ODN prior to beingadded to semi-permeable chambers and irradiated. The vaccine productused the 18-mer IGF-1R AS ODN with the sequence5′-TCCTCCGGAGCCAGACTT-3′, one frameshift downstream from the previoussequence; and, based on its immunostimulatory properties, addition of 2μg of exogenous antisense to each chamber (C-v). The protocol was alsoamended to include a chamber containing PBS (C-p). Up to 10 chamberswere implanted in the rectus sheath. Autologous tumor cell supernatantsobtained during the plating phase of vaccine preparation and explantedchamber contents were flash-frozen for exploratory research objectives.

Study objectives included assessment of safety and radiographicresponses including tumor relative cerebral blood volume (rCBV),apparent diffusion coefficient (ADC), and PET/CT with¹⁸fluorodeoxyglucose dual-time image acquisition at 90 and 240 minutes.Exploratory objectives included serial assessments of peripheral bloodmononuclear cells (PBMC) and chemokines/cytokines in sera, chamberfluids and cell cultures utilizing multiplexed analysis (Luminex).

Immunological Assessments

Plasma leukopheresis was performed one week before surgery for baselineassessment of immune parameters. Blood was also obtainedpost-operatively as previously described¹. Sera and cell fractions wereseparated by centrifugation and cells were treated with red blood celllysis buffer and white blood cells either quantified by flow cytometryor stored in DMSO at −80° C. as were serum samples. Flow cytometry wasperformed as previously described using an EasyCyte 8HT (Millipore) andfluorescently-conjugated mAb specific for human CD4, CD8,CD11b, CD14,CD16, CD20, CD45, CD56, CD80, CD83, and CD 86 (all from BD Biosciences),and CD163 (R&D Systems). Post-collection analysis was performed withFlowJo software (Tree Star Inc, Ashland, Oreg.). Soluble cytokinefactors were quantified using Luminex bead arrays (humancytokine/chemokine panels I, II, and III from Millipore). To assess Tcell polyfunctionality prior to treatment and post operatively, PBMCfrom patients and normal controls were stimulated in vitro with phorbol12-myristate 13-acetate (PMA) and ionomycin (Sigma Aldrich) and thecytokines and chemokines released into culture quantified by Luminex.Tumor tissue sections were assessed by immunohistochemistry for IGF-1R,CD163, CD14, CD206, CD204, CD3, CD4, and CD8. Where possible, Aperioquantification of immunopositive cells was used. Otherwiseimmunostaining was qualitatively assessed by an experiencedneuropathologist (LEK) using an ordinal scale from 0 (no staining) to 6(strong diffuse staining).

Levels of cytokines/chemokines in sera prior to and 2 days followingsurgery, the contents of the explanted chambers as well as supernatantsfrom overnight tumor cell cultures (SN) were all quantitated by Luminex.Membranes from paired vaccine and control chambers were embedded inparaffin for immunohistopathologic examination.

Safety Assessment and Clinical Course

Of 54 severe adverse events recorded, only one SAE was related to theprotocol involving a thrombus from a femoral port used for plasmaleukopheresis. The incidence of DVT in the trial was 8.3%. Nine patientssuccumbed to tumor progression while three patients died from othercauses including intracranial hemorrhage and septicemia (Candidaglabrata, Klebsiella pneumonia). Five autopsies were performed. Allpatients were either weaned off steroids in the post-operative period ormaintained on a daily dose as clinically indicated. Median overallsurvival was 91.4 weeks and correlated highly with the interval betweeninitial surgery and surgery for recurrence. Following recurrent tumorsurgery and autologous cell vaccination two significantly differentprotocol survival cohorts of 48.2 and 10 weeks were identified as longerand short survival cohorts, respectively. (FIG. 19A-FIG. 19C). Excludingone outlier (TJ03), we documented a significant correlation betweenprotocol survival and degree of lymphopenia at enrollment (FIG. 19D).Comparison of values at initial diagnosis and at protocol enrollmentindicated that the mean lymphocyte count had dropped significantly (65%)after standard therapy (eight available paired samples, p=0.012, pairedt-test). There was no significant difference between lymphocyte countsat enrollment and at the last available lymphocyte counts aftervaccination (data not shown).

Radiographic Responses

Anatomic tumor responses were scored and examples are noted in FIG. 20Aand FIG. 20B. Standard MRI anatomic improvements did not correlate withsurvival, but additional imaging criteria did. Three of the four longersurvival cohort (TJ03, TJ06, and TJ09) had a paradoxical increase inrCBV highly correlated with an increasing apparent diffusion coefficient(ADC, see FIG. 20C). This was considered paradoxical because thesepatients, despite perfusion data suggesting disease progression, had ADCvalues reflecting cell loss within the tumor. In the case of TJ06, asignificant and sustained decrease in the CD163+ macrophage populationwas noted at second vaccination that carried forward to autopsy (FIG.20D and see below). In two of these cases PET/CT criteria consistentwith inflammation were observed, corroborating these findings (FIG. 24).Summary cytokine plots favored a pro-inflammatory process in these threepatients (FIG. 20E).

Examination of Explanted Chambers and Pathologic Specimens

Explanted chambers were structurally intact and contained no viablecells by Trypan blue exclusion. Histologic analysis of membranes fromthe chambers revealed that CD15+ neutrophils and CD163+ macrophages werecoating the outer surface of membranes from both C-p and C-v chambersbut with a dramatic increase on C-v (FIG. 3A). The chamber contentsreflected the products of the encapsulated cells and inward diffusion offactors from the surrounding environment, with the control C-p chambercontrolling for the latter. Chemokines elevated in C-v by comparisonwith C-p included CCL21, CCL20, and CCL19, all of which were alsosignificantly increased over serum levels. CXCL12 was elevated in C-vand sera by comparison with C-p (FIG. 25 and Table 6). Also, significantelevations of HSP-70 and granzyme B in C-v compared to sera were noted(3826 pg/ml v. 327 pg/ml, p=0.0015, and 37 pg/ml v. 12 pg/ml, p=0.01,respectively). These results demonstrate that the methods disclosedherein induce pro-inflammatory immune responses that enhance anti-cancereffect.

TABLE 6 Matched pairs analysis of cytokines derived from three sourcesin each of five study subjects. Cytokine (pg/ml) Matched pairscomparison CCl21 C-v 635 C-v > C-p, p = .0385 Source C-p 383 serum < C-v= .0318 (N = 5) serum 214 CCl20 C-v 9430 serum < C-v, p < .0001 SourceC-p 6686 serum < C-p, p < .016 (N = 5) serum 156 CXCL12 C-v 394 C-v >C-p, p = .0224 Source C-p 154 Serum > C-p, p = .012 (N = 5) serum 490C-v vs. serum, NS

Paraffin sections from surgical interventions through autopsies wereavailable for immunohistochemistry. A significant increase in the numberof tumor-infiltrating CD163+ macrophages at vaccination versus initialdiagnosis that decreased significantly at autopsy was noted in allevaluable cases (FIG. 21B and FIG. 26).

Levels of IGF-1R expressing cells were high throughout tumor tissuesfrom initial diagnosis through surgery for recurrence, but a significantdecrease in both at autopsy. Staining was too diffuse for immunopositivecell quantification by Aperio, so a qualitative scale was used.Comparison of pre-vaccination and post-vaccination levels revealed asignificant decrease in IGF-1R positive cells (FIG. 21C and FIG. 26).

Comparing survival cohorts, we noted significantly lower levels ofCD163+ TAMs at both initial diagnosis (3.7% v. 51.5%, p 0.0075) and atvaccination 26% v. 53.9%, p=0.0402) in the long survival compared to theshort survival cohort (FIG. 27). Levels of TAMs correlated highly withcirculating M2 cells in the short survival cohort (FIG. 27B). Few CD3,CD4, or CD8 cells were noted through all serial subject samples (datanot shown).

Chemokine/Cytokine Content in Collected Samples

It was hypothesized that any significant increase incytokines/chemokines in C-v may reflect their elevated presence ineither the tumor microenvironment (TME) or sera. To gain further insightinto this question it was explored whether cytoreductive surgery reducedserum levels of these cytokines. Excluding two outliers, of all serumcytokines/chemokines surveyed, serum CCL21 was significantly lower onpost-operative day 2 (Table 6) supporting CCL21 production from the TME.Of interest, post-vaccination levels of T cells trended closely withlevels of both CCL21 and CXCL12 in the longer survival subjects (FIG.4). In contrast, we noted no associated patterns between T cells,monocytes or cytokines for the short survival cohort (FIG. 28).

CCL2 which was high in both SN and C-v was also significantly elevatedin serum after vaccination suggesting a source of this chemokine otherthan the TME. The mean post-operative serum levels of CCL2 were alsosignificantly higher in the short by comparison with the long survivalcohort (3812 pg/ml vs. 1978 pg/ml, p<0.0078).

After initial vaccination and re-vaccination, the longer survivalsubjects manifested coordinated changes between circulating levels of Tcells, monocytes, and pro-inflammatory chemokines/cytokines. Inverserelationships between T cells and macrophages and between the CD163+subset of circulating CD14+CD16− macrophages and the chemokine CCL2 werenoted. See Table 7 showing certain cytokines. In three of four subjects,circulating levels of CD163+ cells and CCR2+ cells were also directlycorrelated (R²=0.68, p=0.043). A significantly higher CD4/CD8 ratio wasapparent in the longer survival cohort in the post vaccination period.

TABLE 7 Matched pairs of cytokines before and after surgery: cytokineDay −7 Day 2 P value ↓ CCL21 228 120 p < .002 GM-CSF  11.8  6.9 p <.0001 M-CSF  81.5  56.9 p < .034 → CXCL12 499 446 p < .25 MCP-3  24  18p < .09 MDC 257 218 p < .34 CCL20 226 228 p < .512 ↑ CCL2  79.2 476.8 p< .0001 MCP-4  67 189 p < .006 CCL19 133 605 p < .03T Cell Activation In Vitro

PBMC samples obtained at day −7 and day 14 were non-specificallystimulated with PMA/ionomycin and supernatants assessed forchemokine/cytokine levels. After excluding one profoundly lymphopenicoutlier (TJ03), significant differences in the two survival cohorts werenoted for six putative cytokines associated with classical Th-1 and Th-2responses at day 14 (FIG. 5 and Table 8).

TABLE 8 PMA/Ionomycin stimulation before and after surgery, exclusive ofTJ11. Impact of surgery is less for longer v. shorter survival cohorts.Longer survival cohort Short survival cohort Cytokine (pg/ml) (N = 4)mean change (N = 7) mean change IFNγ Day −7 24,859 2,291 35,330 −12,715*Day 14 27,151 p < .5664 22,615 p < .0201 IL2 Day −7 32,589 −10,35029,340  −11,020* Day 14 22,239 p < .0610 18,320 p < .0179 TNFα Day −732,749 −8,487 37,144 −13,805* Day 14 30,522 p < .3875 23,339 p < .0060IL4 Day −7 12,631 −10,287 3,381  −2,708* Day 14 2,344 p < .1559 673 p <.0247 IL5 Day −7 11,340 −9,709 2,080  −1,935* Day 14 1,631 p < .1470 145p < .0383 IL13 Day −7 16,637 −14,511 4,403  −4,010* Day 14 2,126 p <.1605 393 p < .0284 *significance at p < .05.

Example 14: Monocytes Polarized Towards the M2 Cells Overexpress IGF-1R

Immature undifferentiated human monocytes induced to an M2 polarizationby canonical M2 differentiation by IL-4 and IL-13 overexpress IGF-1Rcompared to macrophages induced to an M1 polarization. Further,treatment with IGF-1R AS ODN selectively blocks the appearance ofpolarized M2 cells as well as the survival of existing M2 cells (FIG.29). These observations represent new information about the immunesystem and support a therapeutic intervention involving targetedelimination of the M2 cells associated with poor prognosis in patientswith a variety of cancers. FIG. 29a demonstrates that the vast majorityof IGF-1R AS ODN uptake occurs with monocytes and neutrophils. Despitesimilar uptake of IGF-1R AS ODN in M1 and M2 polarized macrophages,increasing concentrations of IGF-1R AS ODN targets selective eliminationof M2 CD163+ cells with upregulation of IGF-1R only (FIG. 29b ). Therate of apoptotic cell death of CD163+ cells is directly related to theconcentration of IGF-1R AS ODN (FIG. 29c ).

Example 15: Polarization of Monocytes Towards M2 by Incubation of NormalMonocytes in Cancer Patient Sera

An analysis of patients with different types of cancers was performed tosee if their serum was capable of CD163+ differentiation. As shown inFIG. 30, CD163+ macrophage differentiation was noted fromundifferentiated monocytes coincubated with serum from head and necksquamous cell carcinoma (N=2), non-small cell lung carcinoma (N=2), andprostate cancer (N=5). In all cases, treatment with IGF-1R AS ODNknocked this cell population down. This provides confirmation thatfactors present in the sera of patients with a variety of cancers inducepolarization of monocytes towards M2 monocytes differentially expressingCD163 and/or a variety of other phenotypic markers including CD204 andCD206.

Example 16: Monocytes Polarized Towards the M2 CD163+ Phenotype byTreatment with Sera from Patients with Different Cancers ShowUpregulation of Both CD163 and PDL-1

FIG. 31 shows that monocytes polarized towards the M2 CD163+ phenotypeby treatment with sera from patients with different cancers showupregulation of both CD163 as well as PDL-1; in both cases treatmentwith AS ODN knocks down both CD163 and PDL-1 by selectively targetingthis population of cells. FIG. 31A shows a comparison of means for PBScontrol v. IGF-1R AS ODN (NOBEL, 250 μg) treatment of CD163+ macrophagesexpressing PDL-1. FIG. 31B shows that matched pairs analysis revealshighly significant decrease in this cell population reflected assignificant reduction of PDL-1. Removal of a cell population thatover-expresses PDL-1, releases cytotoxic T cells from a source ofinhibition and thereby restores Type 1 immunity in these cancerpatients.

Example 17: Difference in Circulating CD163+ Monocytes Between NormalIndividuals and Astrocytoma Patients

The difference in circulating CD163+ monocytes between normalindividuals and astrocytoma patients was studied. Normal individualshowed ˜6% CD14+ monocytes in their circulation with intermediate levelsof CD163 (FIG. 33A). Two changes are observed in the cancerpatient—higher numbers of monocytes and the monocytes have higher levelsof CD163 (FIG. 33A). Other cells do not have CD163 at all. Normalindividuals can have a wide range of monocytes, due to infections etc.(FIG. 33B, cells positive for CD11b+CD14) but these are elevated inpatients with malignant astrocytomas. The histogram in FIG. 33C showsthat patient monocytes have variably higher levels of CD163 on theirCD14 monocytes than control cells.

Example 18: Tumor-Infiltrating M2 Monocytes and Wildtype IsocitrateDehydrogenase (IDH1) Status are Associated with Gadolinium-Enhancementby MRI and Poor Prognosis in Anaplastic Astrocytoma Patients

Tumor-infiltrating M2 monocytes, wildtype IDH1 status, andgadolinium-enhancement by MRI in anaplastic astrocytoma patients definea more aggressive tumor associated with poor prognosis. Formalin-fixed,paraffin-embedded tissues were stained for the IDHR1 mutation R132H(FIG. 34A) and CD163 (FIG. 34B). Representative images for FLAIR (FIG.34C and FIG. 34D, left panels) and gadolinium-enhanced T1-weighted axialMRI (FIG. 34C and FIG. 34D, right panels) are shown for non-enhancing,AIII (IDH1 R132H mutant grade III) (FIG. 34C) and enhancing, AIII-G(IDH1 wild-type grade III with characteristics of glioblastomamultiforme) (FIG. 34D) tumors. Patients were divided into groups basedon these three aforementioned parameters (FIG. 34A-FIG. 34D),specifically, AIII and AIII-G which resemble more aggressive GBM (FIG.34E, FIG. 34F, and FIG. 34G). Results for the presence (R132H⁺) orabsence (R132H⁻) of the IDH1 mutation in 38 randomly selected MRIenhancing and non-enhancing AA patients are shown in panel FIG. 34E,where n.d. represents none detected. The CD163⁺ cell content in excisedtumor specimens was enumerated using an automated cell counting systemand is presented for AA specimens separated by enhancement in FIG. 34F.Box-and-whisker plots indicate the 75^(th), 50^(th), and 25^(th)percentiles while maximum and minimum data values are represented by theupper and lower whiskers. The statistical significance of the differencebetween the groups was assessed by the Mann Whitney test (***, p<0.001).The Kaplan-Meier survival curves of patients segregated based on theaggressiveness of their tumors are presented in FIG. 34G. Statisticallysignificant survival differences between the groups (**) were determinedby the Log-Rank (p=0.0019) and Wilcoxon tests (p=0.0088). The resultsindicate that IDH R132H mutant grade III astrocytomas rarely enhancewith gadolinium and that, as expected, the accumulation of CD163⁺M2cells in tumor tissues is associated with the loss of vascularintegrity.

Example 19: The Numbers of Circulating Monocytes are Elevated in AIIIand AIII-G Patients and Express Increasing Levels of the M2 Marker CD163

PBMC from 18 randomly selected WHO grade III astrocytoma patients and 24normal donors were stained with antibodies specific for CD11b, CD14, andCD163 and assessed by flow cytometry. Forward scatter (FSC) and sidescatter (SSC) profiles were used to establish a live cell gate andmonocytes were defined as live cells expressing CD11b and CD14 (FIG.35A). Representative contour plots for the live gate and analysis ofCD11b and CD14 positivity in PBMC from a normal and an AA donor areshown in FIG. 35A where axes are presented as log scale and the numbersindicate the frequency of gated cells. FIG. 35B is a summary chartshowing the frequency of CD11b⁺CD14⁺ monocytes in PBMC from 12 patientswith AIII, 6 patients with AIII-G, and 24 normal individuals determinedby flow cytometry. The statistical significance of differences in cellpercentages between normal individuals and AA patient subsets wasassessed by Student's t test (**, p<0.01). The median fluorescenceintensity (MFI) for CD163 staining of CD11b+CD14+ gated monocytes isoverlayed from representative histogram plots of AIII, AIII-G, andnormal blood specimens in FIG. 35C. Axes are presented as log scale. TheMFI for CD163-staining of the gated monocyte subset in PBMC samples fromthe different donor groups are presented in FIG. 35D. Statisticalsignificance was assessed by ANOVA followed by Tukey's post-test (**,p<0.05). While CD11b⁺CD14⁺ monocytes are present at similarly elevatedlevels in the circulation of all of the tested patients, cells from thecirculation of patients with the AIII tumors more closely resembling GBM(Glioblastoma multiforme; AIII-G) express progressively higher levels ofCD163 than those from patients with less malignant AIII tumors andnormal subjects (FIG. 35C and FIG. 35D). As expected due to the increasein circulating monocytes, the frequencies of CD3⁺ and CD20⁺ lymphocytesare decreased in the blood of grade III astrocytoma patients bycomparison with normal individual.

Example 20: Antibodies Present in AIII and AIII-G Patient Serum thatBind Shared Antigens on Astrocytoma Exosomes Differ in Isotype Profile

Exosomes isolated from three astrocytoma patient primary tumor celllines were coated onto 96-well plates and incubated with patient sera(13 AIII, 8 AIII-G) collected before initial surgery and normal controlserum (4). Bound antibodies were detected with fluorescently-conjugatedwhole IgG (FIG. 36A) or secondary antibodies specific for IgG isotypes(FIG. 36B) and the extent of antibody binding measured as MFI. The datais presented as values from individual subjects in box-and-whisker plotsas described in Example 18. The asterisks and bars in FIG. 36A indicatevalues that are significantly different from normal control values asdetermined by ANOVA followed by Dunnett's test (p<0.05). In FIG. 36B,the group of values from AIII-G patients that statistically differedsignificantly from normal control and AIII patient values by ANOVA andTukey's post-test is noted by ** (p=0.004). As shown in FIG. 36A, IgGantibodies reactive with these exosomes are also present in sera fromthe majority of grade III astrocytoma patients regardless of theirprognostic category. However, when isotype specific antibodies were usedfor detection we observed that exosome-binding antibodies of theTh2-associated IgG2 isotype were significantly elevated in AIII-G bycomparison with the longer-lived AIII patients (FIG. 36B). Levels ofIgG1 tended to be slightly elevated in the latter patients while levelsof IgG4 were slightly elevated in AIII-G patients but neither of thesedifferences was significant.

Example 21: Soluble Factors Generally Associated with Th1 and Th2Immunity are Elevated in the Sera of AIII and AIII-G PatientsRespectively

Sera from AA patients segregated based on gadolinium-enhanced MRI intoAIII (n=17) and AIII-G (n=13) subsets was assessed for the levels ofsoluble factors by Luminex. Concentrations for individual specimens arepresented in box-and-whisker plots as described in Example 18. Thestatistical significance of differences between the two groups wasassessed by Student's t test ****, p≤0.001; **, p≤0.01; *, p≤0.05. Serumconcentrations of the Th2 cytokine IL-10 and CCL4 were significantlyelevated in patients with AIII tumors having GBM characteristics whereasIL-9 and the Th1-related IL-12 P40, CXCL10, and FLT3L were significantlyelevated in the remaining AIII patients (FIG. 37).

Example 22: Levels of Expression in PBMC of Genes Encoding White BloodCell Phenotypic Markers, Cytokine and Chemokine Receptors as Well astheir Ligands Differ Between AIII and AIII-G Patients

The copy numbers of genes for monocyte phenotypic markers (FIG. 38A),interleukins (FIG. 38B), interleukin receptors (FIG. 38C), CC chemokines(FIG. 38D) and receptors (FIG. 38E), and CXC chemokines (FIG. 38F) andreceptors (FIG. 38G) in PBMC from 17 unselected AA patients wereassessed by high throughput quantitative RT-PCR and normalized to thecopy numbers of the housekeeping gene L13a present in each sample. LDAwas performed on the normalized copy numbers and is presented in theleft-hand panels where each dot represents data from an individualpatient. Dots representing the results of analysis of individual AIIIand AIII-G patients are colored blue and red respectively. Themultivariate mean for each group is presented as the + at the center ofsimilarly colored circles/ellipses representing the 95% confidenceintervals of the means. Mean copy numbers for each gene tested in thetwo patient cohorts are presented in the accompanying right-hand panelsas heatmaps with red and green corresponding to high versus lowexpression levels respectively and the range of gene copy numbersdetected shown in the associated scale bars. Grey boxes representreactions that failed to generate a detectable product.

Linear discriminant analysis (LDA) was used to determine how well theexpression of each marker class separates and characterizes the twopatient cohorts. FIG. 38A shows the results of LDA for the monocytephenotypic markers CD11b, CD14, CD15, CD68, CD163, CD204, and CD206 withthe corresponding gene expression levels depicted as heat maps. Moderateto strong elevations in the expression levels of mRNAs specific forCD15, CD163 and CD206 (8, 3, 5 times, respectively) in PBMC samples frompatients with AIII-G were observed by comparison with conventional AIIItumors but slightly elevated CD11b and CD204 transcript levels in thelatter (1.9 fold). LDA analysis of the levels of expression of monocytephenotype genes accurately separated 14 of the 17 cases tested into oneof the two patient cohorts. Consistent with the flow cytometry data,CD163 proved to be the most discriminatory phenotypic mRNA marker.Similar LDA performed for 28 interleukin genes correctly classified 100%of the patients into their appropriate cohorts despite the fact thatonly the type 1-associated cytokines IL-15 and IL-32 were significantlydifferent between the AIII and AIII-G samples (p=0.0111 and p=0.0152,respectively), both being lower in the latter (FIG. 38B). Other geneswere either not expressed or expressed at levels that did notsignificantly differ. Analysis of the signatures of 22 interleukinreceptor gene mRNAs in PBMC also clearly discriminated between AIII andAIII-G patients (FIG. 38C). Where differentially expressed, thetranscripts of most of these genes were lower in AIII-G by comparisonwith AIII PBMC. IL-23R and IL31RA in particular were expressed atsignificantly higher levels in the AIII samples (p=0.0055 and p=0.0360,respectively). LDA of the mRNA levels of the 15 out of 21 CCL genesexpressed in PBMC at sufficient levels for this analysis alsodifferentiated the two patient cohorts (FIG. 38D). A trend for elevatedexpression was detected in the AIII samples for CCL3, CCL8, CCL13,CCL21, CCL23 and CCL28 genes, and in the AIII-G samples for CCL2, CCL11and CCL14 genes. However, with the available samples, statisticallysignificant differences were only obtained for CCL3 mRNA, which waspresent at higher levels in AIII by comparison with AIII-G PBMC. LDA ofCCR gene expression data also clearly differentiated the patients (FIG.38E). For most CCR mRNAs, except for CCR4 which was somewhat higher inAIII-G samples, there was a trend for higher expression in theconventional AIII samples, but only two genes, CCR1 and CCR5 weresignificantly overexpressed (p=0.0206 and p=0.0003, respectively). LDAof the CXCL gene expression data accurately characterized 15 of the 17cases tested as belonging to the different patient cohorts despite nostatistically significant differences in mRNA levels (FIG. 38F). Adifference close to significance was detected for only CXCL7 (p=0.0673),which was upregulated in PBMC from AIII-G patients. CXCL2, CXCL10, andCXCL16 transcripts were detected at higher levels in conventional AIIIPBMC but the differences with AIII-G samples were not significant.Comparable results were obtained for LDA based on the expression of CXCchemokine receptors where 14 of the 17 AA cases were accuratelysubgrouped (FIG. 38G). CXCR1, CXCR3, CXCR4, CXCR6, and CX3CR1transcripts were found to be overexpressed in AIII-G, but only CXCR3 andCXCR6 by significant levels (p=0.0111 and p=0.0206, respectively). OnlyCXCR7 mRNA levels were higher in PBMC from patients with conventionalAIII tumors but the difference did not reach statistical significancewith the number of samples analyzed.

Example 23: AIII and AIII-G Patient Subsets can be AccuratelyDifferentiated by the Expression of Select Immunologically-RelevantGenes in PBMC

Discriminant analysis was first used to identify the gene expressiondata, obtained as described in Example 22, that best separated AIII andAIII-G patient PBMC (FIG. 39A). Principal Component Analysis was thenused to determine which of these genes, CCL3, CCR4, CCR5, CCR7, CXCL7,IL-15, IL-32, IL-15R, IL-21R, IL-23R, IL-31RA, and CD163, are mosteffective at differentiating the two patient cohorts (FIG. 39B). Bothdiscriminant and principal component plots were generated usinggene-specific expression profiles for each individual AIII and AIII-GPBMC specimen (represented by dots, colored blue and red, respectively).The dashed green lines in (FIG. 39A) and green vectors in (FIG. 39B)represent the directions of the gene transcripts in the canonical andcomponent spaces respectively. LDA was performed on the mRNA levels ofall 93 genes with detectable signals from PBMC to identify the genesthat most reliably delineate AIII from AIII-G patients (FIG. 39A). Thetwelve genes detailed in Table 9 were selected for PCA analysis (FIG.39B). The total variance explained by the first principal component was37% whereas the second principal component explained nearly 20% of totalvariance. Based on PCA, assessment of the expression levels in PBMC ofthe M2 marker CD163, the proinflammatory cytokine IL-32, and the type 1cytokine receptors IL-21R and IL-23R are sufficient to obtain clearseparation between patients with the different classes of AIII tumors

TABLE 9 Functional characteristics of PBMC-expressed genes selected byDiscriminant Analysis. Gene Immune-related Functions in SymbolFunctional Annotation Cell Expression Circulation CCL3 Inducible,secreted Leukocytes Activation of cell chemotaxis, chemokine ligand fortrafficking and phagocytosis of PBMC CCR1, CCR3, CCR5 by ligand-receptorinteraction CCR4 Chemokine receptor for Th2-, T-reg- cells Activation ofTh2 response by G protein CCL17, CCL22 ligands coupled receptorsignaling CCR5 Chemokine receptor for T-cells, Activation of Th1response by G protein CCL3 CCL4, CCL5, monocytes coupled receptorsignaling CCL8 CCR7 Chemokine receptor for B-, T-, dendritic Homing,migration, induction and CCL19, CCL21 cells maintenance of PRMC by Gprotein coupled receptor signaling CXCL7 Inducible, secreted Platelets,Chemotaxis and activation of neutrophils chemokine ligand formacrophages and macrophages by ligand-receptor CXCR, CXCR2 interactionIL-15 Inducible, secreted Monocytes, Activation of differentiation andcytokine or IL2/IL15 dendritic cells proliferation of PBMC receptorcomplex IL-32 Intracellular and secreted T-, NK-cells Control of PBCsactivation and inducer of inflammatory differentiation cytokines IL-15RCytokine receptor for IL- Monocytes NK, Presentation of IL-15 forintracrine, 15 T, NKT- cells autocrine, paracrine activation of PBMCIL-21R Cytokine receptor for IL- B, T, NK-cells Activation of PBMC byligand-21R/IL- 21 2R, IL-21R/IL-7R, IL-21R/IL-15RA interaction IL-23RCytokine receptor for IL- Leukocytes Activation of T, NK and dendriticcells 23 by ligand-IL-23R/IL-12RB1 interaction IL-31RA Cytokine receptorfor IL- Monocytes, T- Receptor signaling via STAT-3, STAT-5 31 cellsactivation in monocytes and T-cell subsets CD163 Scavenger receptorMonocytes, Regulation of Th2-cell differentiation, macrophages clearanceof substances by receptor mediated endocytosis

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosure isnot entitled to antedate such publication by virtue of prior disclosure.

What is claimed is:
 1. A method for treating cancer in a subject in needthereof, the method comprising: systemically administering to thesubject an effective amount of a pharmaceutical composition comprising afirst Insulin-like Growth Factor-1 Receptor antisense oligonucleotide(IGF-1R AS ODN), wherein the sequence of the first IGF-1R AS ODN is SEQID NO: 1, and wherein the first IGF-1R AS ODN has a modified phosphatebackbone; and administering to the subject at least one biodiffusionchamber, the chamber comprising tumor cells and a second IGF-1R AS ODN,wherein the sequence of the second IGF-1R AS ODN is SEQ ID NO:1, andwherein the second IGF-1R AS ODN has a modified phosphate backbone. 2.The method of claim 1, wherein the biodiffusion chamber is administeredto the subject prior to systemically administering the pharmaceuticalcomposition.
 3. The method of claim 1, wherein the biodiffusion chamberis administered to the subject subsequent to systemically administeringthe pharmaceutical composition.
 4. The method of claim 1, wherein thetumor cells and the second IGF-1R AS ODN are each irradiated.
 5. Themethod of claim 1, wherein the biodiffusion chamber is surgicallyimplanted into the rectus sheath of the subject for a therapeuticallyeffective time.
 6. The method of claim 1, wherein the modified phosphatebackbone of the first IGF-1R AS ODN renders the first IGF-1R AS ODNresistant to nuclease degradation.
 7. The method of claim 6, wherein themodified phosphate backbone of the first IGF-1R AS ODN comprises atleast one phosphorothioate linkage.
 8. The method of claim 6, whereinthe modified phosphate backbone of the first IGF-1R AS ODN comprises upto about 50% phosphorothioate-linkages.
 9. The method of claim 6,wherein the modified phosphate backbone of the first IGF-1R AS ODN isfully phosphorothioate-linked.
 10. The method of claim 5, wherein thefirst IGF-1R AS ODN has at least one terminal modification.
 11. Themethod of claim 10, wherein the at least one terminal modification is aphosphorothioate monophosphate.
 12. The method of claim 10, the at leastone terminal modification comprises a 5′ cap structure and/or a 3′ capstructure.
 13. The method of claim 1, wherein the modified phosphatebackbone of the second IGF-1R AS ODN renders the second IGF-1R AS ODNresistant to nuclease degradation.
 14. The method of claim 13, whereinthe modified phosphate backbone of the second IGF-1R AS ODN comprises atleast one phosphorothioate linkage.
 15. The method of claim 14, whereinthe modified phosphate backbone of the second IGF-1R AS ODN comprises upto about 50% phosphorothioate linkages.
 16. The method of claim 14,wherein the modified phosphate backbone of the second IGF-1R AS ODN isfully phosphorothioate-linked.
 17. The method of claim 13, wherein thesecond IGF-1R AS ODN has at least one terminal modification.
 18. Themethod of claim 17, wherein the at least one terminal modification is aphosphorothioate monophosphate.
 19. The method of claim 17, the at leastone terminal modification comprises a 5′ cap structure and/or a 3′ capstructure.
 20. The method of claim 1, wherein the modified phosphatebackbone of the first IGF-1R AS ODN and the modified phosphate backboneof the second IGF-1R AS ODN are the same.
 21. The method of claim 1,wherein the modified phosphate backbone of the first IGF-1R AS ODN andthe modified phosphate backbone of the second IGF-1R AS ODN aredifferent.
 22. The method of claim 1, wherein the cancer is selectedfrom the group consisting of glioma, astrocytoma, breast cancer, headand neck squamous cell cancer, papillary renal cell carcinoma Type II,lung cancer, pancreatic cancer, gall bladder cancer, rectal cancer,classical Hodgkin's lymphoma, ovarian cancer, and colorectal cancer. 23.The method of claim 22, wherein the cancer is glioma.